Synthesis of long-chain polyunsaturated fatty acids by recombinant cell

ABSTRACT

The present invention relates to methods of synthesizing long-chain polyunsaturated fatty acids, especially eicosapentaenoic acid, docosapentaenoic acid and docosahexaenoic acid, in recombinant cells such as yeast or plant cells. Also provided are recombinant cells or plants which produce long-chain polyunsaturated fatty acids. Furthermore, the present invention relates to a group of new enzymes which possess desaturase or elongase activity that can be used in methods of synthesizing long-chain polyunsaturated fatty acids.

This application is a continuation of U.S. Ser. No. 13/243,747, filedSep. 23, 2011, a continuation of U.S. Ser. No. 12/661,978, filed Mar.26, 2010, now U.S. Pat. No. 8,106,226, issued Jan. 31, 2012, acontinuation of U.S. Ser. No. 11/112,882, filed Apr. 22, 2005, now U.S.Pat. No. 7,807,849, issued Oct. 5, 2010 which claims the benefit of U.S.Provisional Application Nos. 60/668,705, filed Apr. 5, 2005; 60/613,861,filed Sep. 27, 2004; and 60/564,627, filed Apr. 22, 2004; and claimspriority of Australian Provisional Application No. 2005901673, filedApr. 5, 2005, the content of all of which are hereby incorporated byreference into the subject application.

FIELD OF THE INVENTION

The present invention relates to methods of synthesizing long-chainpolyunsaturated fatty acids, especially eicosapentaenoic acid,docosapentaenoic acid and docosahexaenoic acid, in recombinant cellssuch as yeast or plant cells. Also provided are recombinant cells orplants which produce long-chain polyunsaturated fatty acids.Furthermore, the present invention relates to a group of new enzymeswhich possess desaturase or elongase activity that can be used inmethods of synthesizing long-chain polyunsaturated fatty acids.

BACKGROUND OF THE INVENTION

Omega-3 long-chain polyunsaturated fatty acid(s) (LC-PUFA) are nowwidely recognized as important compounds for human and animal health.These fatty acids may be obtained from dietary sources or by conversionof linoleic (LA, omega-6) or α-linolenic (ALA, omega-3) fatty acids,both of which are regarded as essential fatty acids in the human diet.While humans and many other vertebrate animals are able to convert LA orALA, obtained from plant sources, to LC-PUFA, they carry out thisconversion at a very low rate. Moreover, most modern societies haveimbalanced diets in which at least 90% of polyunsaturated fatty acid(s)(PUFA) consist of omega-6 fatty acids, instead of the 4:1 ratio or lessfor omega-6:omega-3 fatty acids that is regarded as ideal (Trautwein,2001). The immediate dietary source of LC-PUFA such as eicosapentaenoicacid (EPA, 20:5) and docosahexaenoic acid (DHA, 22:6) for humans ismostly from fish or fish oil. Health professionals have thereforerecommended the regular inclusion of fish containing significant levelsof LC-PUFA into the human diet. Increasingly, fish-derived LC-PUFA oilsare being incorporated into food products and in infant formula.However, due to a decline in global and national fisheries, alternativesources of these beneficial health-enhancing oils are needed.

Inclusion of omega-3 LC-PUFA such as EPA and DHA in the human diet hasbeen linked with numerous health-related benefits. These includeprevention or reduction of coronary heart disease, hypertension, type-2diabetes, renal disease, rheumatoid arthritis, ulcerative colitis andchronic obstructive pulmonary disease, and aiding brain development andgrowth (Simopoulos, 2000). More recently, a number of studies have alsoindicated that omega-3 PUFA may be beneficial in infant nutrition anddevelopment and against various mental disorders such as schizophrenia,attention deficit hyperactive disorder and Alzheimer's disease.

Higher plants, in contrast to animals, lack the capacity to synthesisepolyunsaturated fatty acids with chain lengths longer than 18 carbons.In particular, crop and horticultural plants along with otherangiosperms do not have the enzymes needed to synthesize the longerchain omega-3 fatty acids such as EPA, DPA and DHA that are derived fromALA. An important goal in plant biotechnology is therefore theengineering of crop plants, particularly oilseed crops, that producesubstantial quantities of LC-PUFA, thus providing an alternative sourceof these compounds.

Pathways of LC-PUFA Synthesis

Biosynthesis of LC-PUFA from linoleic and α-linolenic fatty acids inorganisms such as microalgae, mosses and fungi may occur by a series ofalternating oxygen-dependent desaturations and elongation reactions asshown schematically in FIG. 1. In one pathway (FIG. 1, II), thedesaturation reactions are catalysed by Δ6, Δ5, and Δ4 desaturases, eachof which adds an additional double bond into the fatty acid carbonchain, while each of a Δ6 and a Δ5 elongase reaction adds a two-carbonunit to lengthen the chain. The conversion of ALA to DHA in theseorganisms therefore requires three desaturations and two elongations.Genes encoding the enzymes required for the production of DHA in thisaerobic pathway have been cloned from various microorganisms and lowerplants including microalgae, mosses, fungi. Genes encoding some of theenzymes including one that catalyses the fifth step, the Δ5 elongase,have been isolated from vertebrate animals including mammals (reviewedin Sayanova and Napier, 2004). However, the Δ5 elongase isolated fromhuman cells is not specific for the EPA to DPA reaction, having a widespecificity for fatty acid substrates (Leonard et al., 2002).

Alternative routes have been shown to exist for two sections of the ALAto DHA pathway in some groups of organisms. The conversion of ALA to ETAmay be carried out by a combination of a Δ9 elongase and a Δ8 desaturase(the so-called Δ8 desaturation route, see FIG. 1, IV) in certainprotists and thraustochytrids, as evidenced by the isolated of genesencoding such enzymes (Wallis and Browse, 1999; Qi et al., 2002). Inmammals, the so-called “Sprecher” pathway converts DPA to DHA by threereactions, independent of a Δ4 desaturase (Sprecher et al., 1995).

Besides these desaturase/elongase systems, EPA and DHA can also besynthesized through an anaerobic pathway in a number of organisms suchas Shewanella, Mortiella and Schizhochytrium (Abbadi et al., 2001). Theoperons encoding these polyketide synthase (PKS) enzyme complexes havebeen cloned from some bacteria (Morita et al., 2000; Metz et al., 2001;Tanaka et al. 1999; Yazawa, 1996; Yu et al., 2000; WO 00/42195). The EPAPKS operon isolated from Shewanella spp has been expressed inSynechococcus allowing it to synthesize EPA (Takeyama et al., 1997). Thegenes encoding these enzymes are arranged in relatively large operons,and their expression in transgenic plants has not been reported.Therefore it remains to be seen if the anaerobic PKS-like system is apossible alternative to the more classic aerobic desaturase/elongase forthe transgenic synthesis of LC-PUFA.

Desaturases

The desaturase enzymes that have been shown to participate in LC-PUFAbiosynthesis all belong to the group of so-called “front-end”desaturases which are characterised by the presence of a cytochrome b₅domain at the N-terminus of each protein. The cyt b₅ domain presumablyacts as a receptor of electrons required for desaturation (Napier etal., 1999; Sperling and Heinz, 2001).

The enzyme Δ5 desaturase catalyses the further desaturation of C20LC-PUFA leading to arachidonic acid (ARA, 20:40ω6) and EPA (20:5ω3).Genes encoding this enzyme have been isolated from a number oforganisms, including algae (Thraustochytrium sp. Qiu et al., 2001),fungi (M. alpine, Pythium irregulare, Michaelson et al., 1998; Hong etal., 2002), Caenorhabditis elegans and mammals. A gene encoding abifunctional Δ5-/Δ6-desaturase has also been identified from zebrafish(Hasting et al., 2001). The gene encoding this enzyme might represent anancestral form of the “front-end desaturase” which later duplicated andevolved distinct functions. The last desaturation step to produce DHA iscatalysed by a Δ4 desaturase and a gene encoding this enzyme has beenisolated from the freshwater protist species Euglena gracilis and themarine species Thraustochytrium sp. (Qiu et al., 2001; Meyer et al.,2003).

Elongases

Several genes encoding PUFA-elongation enzymes have also been isolated(Sayanova and Napier, 2004). The members of this gene family wereunrelated to the elongase genes present in higher plants, such as FAE1of Arabidopsis, that are involved in the extension of saturated andmonounsaturated fatty acids. An example of the latter is erucic acid(22:1) in Brassicas. In some protist species, LC-PUFA are synthesized byelongation of linoleic or α-linolenic acid with a C2 unit, beforedesaturation with Δ8 desaturase (FIG. 1 part IV; “Δ8-desaturation”pathway). Δ6 desaturase and Δ6 elongase activities were not detected inthese species. Instead, a Δ9-elongase activity would be expected in suchorganisms, and in support of this, a C18 Δ9-elongase gene has recentlybeen isolated from Isochrysis galbana (Qi et al., 2002).

Engineered Production of LC-PUFA

Transgenic oilseed crops that are engineered to produce major LC-PUFA bythe insertion of these genes have been suggested as a sustainable sourceof nutritionally important fatty acids. However, the requirement forcoordinate expression and activity of five new enzymes encoded by genesfrom possibly diverse sources has made this goal difficult to achieveand the proposal remained speculative until now.

The LC-PUFA oxygen-dependent biosynthetic pathway to form EPA (FIG. 1)has been successfully constituted in yeast by the co-expression of aΔ6-elongase with Δ6- and Δ5 fatty acid desaturases, resulting in smallbut significant accumulation of ARA and EPA from exogenously suppliedlinoleic and α-linolenic acids (Beaudoin et al., 2000; Zank et al.,2000). This demonstrated the ability of the genes belonging to theLC-PUFA synthesis pathway to function in heterologous organisms.However, the efficiency of producing EPA was very low. For example,three genes obtained from C. elegans, Borago officinalis and Mortierellaalpina were expressed in yeast (Beaudoin et al., 2000). When thetransformed yeast were supplied with 18:2ω-3 (LA) or 18:3ω-3 (ALA),there was slight production of 20:4ω-6 or 20:5ω-3, at conversionefficiencies of 0.65% and 0.3%, respectively. Other workers similarlyobtained very low efficiency production of EPA by using genes expressingtwo desaturases and one elongase in yeast (Domergue et al., 2003a; Zanket al., 2002). There remains, therefore, a need to improve theefficiency of production of EPA in organisms such as yeast, let alonethe production of the C22 PUFA which requires the provision ofadditional enzymatic steps.

Some progress has been made in the quest for introducing the aerobicLC-PUFA biosynthetic pathway into higher plants including oilseed crops(reviewed by Sayanova and Napier, 2004; Drexler et al., 2003; Abbadi etal., 2001). A gene encoding a Δ6-fatty acid desaturase isolated fromborage (Borago officinalis) was expressed in transgenic tobacco andArabidopsis, resulting in the production of GLA (18:3ω6) and SDA(18:4ω3), the direct precursors for LC-PUFA, in the transgenic plants(Sayanova et al., 1997; 1999). However, this provides only a single,first step.

Domergue et al. (2003a) used a combination of three genes, encoding Δ6-and Δ5 fatty acid desaturases and a Δ6-elongase in both yeast andtransgenic linseed. The desaturase genes were obtained from the diatomPhaeodactylum tricornutum and the elongase gene from the mossPhyscomitrella patens. Low elongation yields were obtained forendogenously produced Δ6-fatty acids in yeast cells (i.e. combining thefirst and second enzymatic steps), and the main C20 PUFA product formedwas 20:2^(Δ11,14), representing an unwanted side reaction. Domergue etal. (2003a) also state, without presenting data, that the combination ofthe three genes were expressed in transgenic linseed which consequentlyproduced ARA and EPA, but that production was inefficient. Theycommented that the same problem as had been observed in yeast existed inthe seeds of higher plants and that the “bottleneck” needed to becircumvented for production of LC-PUFA in oil seed crops.

WO 2004/071467 (DuPont) reported the expression of various desaturasesand elongases in soybean cells but did not show the synthesis of DHA inregenerated plants or in seeds.

Abbadi et al. (2004) described attempts to express combinations ofdesaturases and elongases in transgenic linseed, but achieved only lowlevels of synthesis of EPA. Abbadi et al. (2004) indicated that theirlow levels of EPA production were also due to an unknown “bottleneck”.

Qi et al. (2004) achieved synthesis in leaves but did not report resultsin seeds. This is an important issue as the nature of LC-PUFA synthesiscan vary between leaves and seeds. In particular, oilseeds store lipidin seeds mostly as TAG while leaves synthesize the lipid mostly asphosphatidyl lipids. Furthermore, Qi et al. (2004) only produced AA andEPA.

As a result, there is a need for further methods of producing long-chainpolyunsaturated, particularly EPA, DPA and DHA, in recombinant cells.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a recombinant cellwhich is capable of synthesising a long chain polyunsaturated fattyacid(s) (LC-PUFA), comprising one or more polynucleotides which encodeat least two enzymes each of which is a Δ5/Δ6 bifunctional desaturase,Δ5 desaturase, Δ6 desaturase, Δ5/Δ6 bifunctional elongase, Δ5 elongase,Δ6 elongase, Δ4 desaturase, Δ9 elongase, or Δ8 desaturase, wherein theone or more polynucleotides are operably linked to one or more promotersthat are capable of directing expression of said polynucleotides in thecell, wherein said recombinant cell is derived from a cell that is notcapable of synthesising said LC-PUFA.

In a second aspect, the present invention provides a recombinant cellwith an enhanced capacity to synthesize a LC-PUFA relative to anisogenic non-recombinant cell, comprising one or more polynucleotideswhich encode at least two enzymes each of which is a Δ5/Δ6 bifunctionaldesaturase, Δ5 desaturase, 46 desaturase, Δ5/Δ6 bifunctional elongase,Δ5 elongase, Δ6 elongase, Δ4 desaturase, Δ9 elongase, or Δ8 desaturase,wherein the one or more polynucleotides are operably linked to one ormore promoters that are capable of expressing said polynucleotides insaid recombinant cell.

In one embodiment, at least one of the enzymes is a Δ5 elongase.

The present inventors are the first to identify an enzyme which hasgreater Δ5 elongase activity than Δ6 elongase activity. As a result,this enzyme provides an efficient means of producing DPA in arecombinant cell as the Δ5 elongation of EPA is favoured over the Δ6elongation of SDA. Thus, in an embodiment, the Δ5 elongase is relativelyspecific, that is, where the Δ5 elongase also has Δ6 elongase activitythe elongase is more efficient at synthesizing DPA from EPA than it isat synthesizing ETA from SDA.

In another embodiment, the Δ5 elongase comprises

i) an amino acid sequence as provided in SEQ ID NO:2,

ii) an amino acid sequence which is at least 50%, more preferably atleast 80%, even more preferably at least 90%, identical to SEQ ID NO:2,or

iii) a biologically active fragment of i) or ii).

In another embodiment, the Δ5 elongase can be purified from algae.

In another embodiment, at least one of the enzymes is a Δ9 elongase.

The present inventors are the first to identify an enzyme which has bothΔ9 elongase activity and Δ6 elongase activity. When expressed in a cellwith a Δ6 desaturase and a Δ8 desaturase this enzyme can use the twoavailable pathways to produce ETA from ALA. DGLA from LA, or both (seeFIG. 1), thus increasing the efficiency of ETA and/or DGLA production.Thus, in an embodiment, the Δ9 elongase also has Δ6 elongase activity.Preferably, the Δ9 elongase is more efficient at synthesizing ETrA fromALA than it is at synthesizing ETA from SDA. Furthermore, in anotherembodiment the Δ9 elongase is able to elongate SDA to ETA, GLA to DGLA,or both, in a yeast cell.

In a further embodiment, the Δ9 elongase comprises

i) an amino acid sequence as provided in SEQ ID NO:3, SEQ ID NO:85 orSEQ ID NO:86,

ii) an amino acid sequence which is at least 50%, more preferably atleast 80%, even more preferably at least 90%, identical to SEQ ID NO:3,SEQ ID NO:85 or SEQ ID NO:86, or

iii) a biologically active fragment of i) or ii).

Preferably, the Δ9 elongase can be purified from algae or fungi.

It is well known in the art that the greater the number of transgenes inan organism, the greater the likelihood that at least one fitnessparameter of the organism, such as expression level of at least one ofthe transgenes, growth rate, oil production, reproductive capacity etc,will be compromised. Accordingly, it is desirable to minimize the numberof transgenes in a recombinant cell. To this end, the present inventorshave devised numerous strategies for producing LC-PUFA's in a cell whichavoid the need for a gene to each step in the relevant pathway.

Thus, in another embodiment, at least one of the enzymes is a Δ5/Δ6bifunctional desaturase or a Δ5/Δ6 bifunctional elongase. The Δ5/Δ6bifunctional desaturase may be naturally produced by a freshwaterspecies of fish.

In a particular embodiment, the Δ5/Δ6 bifunctional desaturase comprises

i) an amino acid sequence as provided in SEQ ID NO:15,

ii) an amino acid sequence which is at least 50%, more preferably atleast 80%, even more preferably at least 90%, identical to SEQ ID NO:15,or

iii) a biologically active fragment of i) or ii).

Preferably, the Δ5/Δ6 bifunctional desaturase is naturally produced by afreshwater species of fish.

Preferably, the Δ5/Δ6 bifunctional elongase comprises

i) an amino acid sequence as provided in SEQ ID NO:2 or SEQ ID NO:14,

ii) an amino acid sequence which is at least 50%, more preferably atleast 80%, even more preferably at least 90%, identical to SEQ ID NO:2or SEQ ID NO:14, or

iii) a biologically active fragment of i) or ii).

In another embodiment, at least one of the enzymes is a Δ5 desaturase.

In a further embodiment, at least one of the enzymes is a Δ8 desaturase.

In another embodiment, the LC-PUFA is docosahexaenoic acid (DHA).

Preferably, the introduced polynucleotide(s) encode three or fourenzymes each of which is a Δ5/Δ6 bifunctional desaturase, Δ5 desaturase,Δ6 desaturase, Δ5/Δ6 bifunctional elongase, Δ5 elongase, Δ6 elongase, orΔ4 desaturase. More preferably, the enzymes are any one of the followingcombinations;

i) a Δ5/Δ6 bifunctional desaturase, a Δ5/Δ6 bifunctional elongase, and aΔ4 desaturase,

ii) a Δ5/Δ6 bifunctional desaturase, a Δ5 elongase, a Δ6 elongase, and aΔ4 desaturase, or

iii) a Δ5 desaturase, a Δ6 desaturase, a Δ5/Δ6 bifunctional elongase,and a Δ4 desaturase.

In another embodiment, the LC-PUFA is DHA and the introducedpolynucleotide(s) encode five enzymes wherein the enzymes are any one ofthe following combinations;

i) a Δ4 desaturase, a Δ5 desaturase, a Δ6 desaturase, a Δ5 elongase anda Δ6 elongase, or

ii) a Δ4 desaturase, a Δ5 desaturase, a Δ8 desaturase, a Δ5 elongase anda Δ9 elongase.

In a further embodiment, the cell is of an organism suitable forfermentation, and the enzymes are at least a Δ5/Δ6 bifunctionaldesaturase, a Δ5 elongase, a Δ6 elongase, and a Δ4 desaturase.

In another embodiment, the LC-PUFA is docosapentaenoic acid (DPA).Preferably, the introduced polynucleotide(s) encode two or three enzymeseach of which is a Δ5/Δ6 bifunctional desaturase, Δ5 desaturase, Δ6desaturase, Δ5/Δ6 bifunctional elongase. Δ5 elongase, or Δ6 elongase.More preferably, the enzymes are any one of the following combinations;

i) a Δ5/Δ6 bifunctional desaturase and a Δ5/Δ6 bifunctional elongase,

ii) a Δ5/Δ6 bifunctional desaturase, a Δ5/Δ6 elongase, and a Δ6elongase, or

iii) a Δ5 desaturase, a Δ6 desaturase, and a Δ5/Δ6 bifunctionalelongase.

In a further embodiment, the LC-PUFA is DPA and the introducedpolynucleotide(s) encode four enzymes wherein the enzymes are any one ofthe following combinations:

i) a Δ5 desaturase, a Δ6 desaturase, a Δ5 elongase and a Δ6 elongase, or

ii) a Δ5 desaturase, a Δ8 desaturase, a Δ5 elongase and a Δ9 elongase.

In another embodiment, the cell is of an organism suitable forfermentation, and the enzymes are at least a Δ5/Δ6 bifunctionaldesaturase, a Δ5 elongase, and a Δ6 elongase.

In a further embodiment, the LC-PUFA is eicosapentaenoic acid (EPA).

Preferably, the introduced polynucleotide(s) encode a Δ5/Δ6 bifunctionaldesaturase and a Δ5/Δ6 bifunctional elongase.

In another embodiment, the introduced polynucleotide(s) encode threeenzymes wherein the enzymes are any one of the following combinations;

i) a Δ5 desaturase, a Δ6 desaturase, and a Δ6 elongase, or

ii) a Δ5 desaturase, a Δ8 desaturase, and a Δ9 elongase.

Evidence to date suggests that desaturases expressed in at least somerecombinant cells, particularly yeast, have relatively low activity.However, the present inventors have identified that this may be afunction of the capacity of the desaturase to use acyl-CoA as asubstrate in LC-PUFA synthesis. In this regard, it has also beendetermined that desaturase of vertebrate origin are particularly usefulfor the production of LC-PUFA in recombinant cells, for example, plantcells, seeds, or yeast. Thus, in another preferred embodiment, therecombinant cell comprises either

i) at least one Δ5 elongase catalyses the conversion of EPA to DPA inthe cell,

ii) at least one desaturase which is able to act on an acyl-CoAsubstrate,

iii) at least one desaturase from a vertebrate or a variant desaturasethereof, or

iv) any combination of i), ii) or iii).

In a particular embodiment, the Δ5 elongase comprises

i) an amino acid sequence as provided in SEQ ID NO:2,

ii) an amino acid sequence which is at least 50% identical to SEQ IDNO:2, or

iii) a biologically active fragment of i) or ii).

The desaturase able to act on an acyl-CoA substrate or from a vertebratemay be a Δ5 desaturase, a Δ6 desaturase, or both. In a particularembodiment, the desaturase comprises

i) an amino acid sequence as provided in SEQ ID NO:16, SEQ ID NO:21 orSEQ ID NO:22,

ii) an amino acid sequence which is at least 50% identical to SEQ IDNO:16, SEQ ID NO:21 or SEQ ID NO:22, or

iii) a biologically active fragment of i) or ii).

Preferably, the at least one desaturase is naturally produced by avertebrate.

Alternatively, when the cell is a yeast cell, the LC-PUFA is DHA, andthe enzymes are at least a Δ5/Δ6 bifunctional desaturase, a Δ5 elongase,a Δ6 elongase, and a Δ4 desaturase.

In a further alternative, when the cell is a yeast cell, the LC-PUFA isDPA, and the enzymes are at least a Δ5/Δ6 bifunctional desaturase, a Δ5elongase, and a Δ6 elongase.

Although the cell may be any cell type, preferably, said cell is capableof producing said LC-PUFA from endogenously produced linoleic acid (LA),α-linolenic acid (ALA), or both. More preferably, the ratio of theendogenously produced ALA to LA is at least 1:1 or at least 2:1.

In one embodiment, the cell is a plant cell, a plant cell from anangiosperm, an oilseed plant cell, or a cell in a seed. Preferably, atleast one promoter is a seed specific promoter.

In another embodiment, the cell is of a unicellular microorganism.Preferably, the unicellular microorganism is suitable for fermentation.Preferably, the microorganism is a yeast.

In a further embodiment, the cell is a non-human animal cell or a humancell in vitro.

In a further embodiment, the recombinant cell produces a LC-PUFA whichis incorporated into triacylglycerols in said cell. More preferably, atleast 50% of the LC-PUFA that is produced in said cell is incorporatedinto triacylglycerols.

In another embodiment, at least the protein coding region of one, two ormore of the polynucleotides is obtained from an algal gene. Preferably,the algal gene is from the genus Pavlova such as from the speciesPavlova salina.

In another aspect, the present invention provides a recombinant cellthat is capable of producing DHA from a fatty acid which is ALA, LA,GLA, ARA, SDA, ETA, EPA, or any combination or mixture of these, whereinsaid recombinant cell is derived from a cell that is not capable ofsynthesising DHA.

In a further aspect, the present invention provides a recombinant cellthat is capable of producing DPA from a fatty acid which is ALA, LA,GLA, ARA, SDA, ETA, EPA, or any combination or mixture of these, whereinsaid recombinant cell is derived from a cell that is not capable ofsynthesising DPA.

In yet a further aspect, the present invention provides a recombinantcell that is capable of producing EPA from a fatty acid which is ALA,LA, GLA, SDA, ETA or any combination or mixture of these, wherein saidrecombinant cell is derived from a cell that is not capable ofsynthesising EPA.

In another aspect, the present invention provides a recombinant cellthat is capable of producing both ETrA from ALA and ETA from SDA, andwhich produces EPA from a fatty acid which is ALA, LA, GLA, SDA, ETA, orany combination or mixture of these, wherein said recombinant cell isderived from a cell that is not capable of synthesising ETrA, ETA orboth.

In a further aspect, the present invention provides a recombinant cellof an organism useful in fermentation processes, wherein the cell iscapable of producing DPA from LA. ALA, arachidonic acid (ARA),eicosatetraenoic acid (ETA), or any combination or mixture of these,wherein said recombinant cell is derived from a cell that is not capableof synthesising DPA.

In another aspect, the present invention provides a recombinant plantcell capable of producing DPA from LA, ALA, EPA, or any combination ormixture of these, wherein the plant cell is from an angiosperm.

In an embodiment, the plant cell is also capable of producing DHA.

In yet another aspect, the present invention provides a recombinant cellwhich is capable of synthesising DGLA, comprising a polynucleotide(s)encoding one or both of:

a) a polypeptide which is an Δ9 elongase, wherein the Δ9 elongase isselected from the group consisting of:

-   -   i) a polypeptide comprising an amino acid sequence as provided        in SEQ ID NO:3, SEQ ID NO:85 or SEQ ID NO:86,    -   ii) a polypeptide comprising an amino acid sequence which is at        least 40% identical to SEQ ID NO:3, SEQ ID NO:85 or SEQ ID        NO:86, and    -   iii) a biologically active fragment of i) or ii), and/or

b) a polypeptide which is an Δ8 desaturase, wherein the Δ8 desaturase isselected from the group consisting of:

-   -   i) a polypeptide comprising an amino acid sequence as provided        in SEQ ID NO:1,    -   ii) a polypeptide comprising an amino acid sequence which is at        least 40% identical to SEQ ID NO:1, and    -   iii) a biologically active fragment of i) or ii),

wherein the polynucleotide(s) is operably linked to one or morepromoters that are capable of directing expression of saidpolynucleotide(s) in the cell, and wherein said recombinant cell isderived from a cell that is not capable of synthesising DGLA.

In an embodiment, the cell is capable of converting DGLA to ARA.

In another embodiment, the cell further comprises a polynucleotide whichencodes a Δ5 desaturase, wherein the polynucleotide encoding the Δ5desaturase is operably linked to one or more promoters that are capableof directing expression of said polynucleotide in the cell, and whereinthe cell is capable of producing ARA.

In a particular embodiment, the cell lacks ω3 desaturase activity and isnot capable of producing ALA. Such cells may be naturally occurring, orproduced by reducing the ω3 desaturase activity of the cell usingtechniques well known in the art.

Preferably, the cell is a plant cell or a cell of an organism suitablefor fermentation.

In a further embodiment, a recombinant cell of the invention alsopossesses the enzyme required to perform the “Sprecher” pathway ofconverting EPA to DHA. These enzymes may be native to the cell orproduced recombinantly. Such enzymes at least include a Δ7 elongase, Δ6desaturase and enzymes required for the peroxisomal β-oxidation oftetracosahexaenoic acid to produce DHA.

The present inventors have also identified a group of new desaturasesand elongases. As a result, further aspects of the invention relate tothese enzymes, as well as homologs/variants/derivatives thereof.

The polypeptide may be a fusion protein further comprising at least oneother polypeptide sequence.

The at least one other polypeptide may be a polypeptide that enhancesthe stability of a polypeptide of the present invention, or apolypeptide that assists in the purification of the fusion protein.

Also provided are isolated polynucleotides which, inter alia, encodepolypeptides of the invention.

In a further aspect, the present invention provides a vector comprisingor encoding a polynucleotide according to the invention. Preferably, thepolynucleotide is operably linked to a seed specific promoter.

In another aspect, the present invention provides a recombinant cellcomprising an isolated polynucleotide according to the invention.

In a further aspect, the present invention provides a method ofproducing a cell capable of synthesising one or more LC-PUFA, the methodcomprising introducing into the cell one or more polynucleotides whichencode at least two enzymes each of which is a Δ5/Δ6 bifunctionaldesaturase, Δ5 desaturase, Δ6 desaturase, Δ5/Δ6 bifunctional elongase,Δ5 elongase, Δ6 elongase, Δ4 desaturase, Δ9 elongase, or Δ8 desaturase,wherein the one or more polynucleotides are operably linked to one ormore promoters that are capable of directing expression of saidpolynucleotides in the cell.

In another aspect, the present invention provides a method of producinga recombinant cell with an enhanced capacity to synthesize one or moreLC-PUFA, the method comprising introducing into a first cell one or morepolynucleotides which encode at least two enzymes each of which is aΔ5/Δ6 bifunctional desaturase, Δ5 desaturase, Δ6 desaturase, Δ5/Δ6bifunctional elongase, Δ5 elongase, Δ6 elongase, Δ4 desaturase, Δ9elongase, or Δ8 desaturase, wherein the one or more polynucleotides areoperably linked to one or more promoters that are capable of directingexpression of said polynucleotides in the recombinant cell, and whereinsaid recombinant cell has an enhanced capacity to synthesize said one ormore LC-PUFA relative to said first cell.

Naturally, it will be appreciated that each of the embodiments describedherein in relation to the recombinant cells of the invention willequally apply to methods for the production of said cells.

In a further aspect, the present invention provides a cell produced by amethod of the invention.

In another aspect, the present invention provides a transgenic plantcomprising at least one recombinant cell according to the invention.

Preferably, the plant is an angiosperm. More preferably, the plant is anoilseed plant.

In a further embodiment, the transgenic plant, or part thereof includinga transgenic seed, does not comprise a transgene which encodes an enzymewhich preferentially converts an ω6 LC-PUFA into an ω3 LC-PUFA.

In yet a further embodiment, the transgenic plant, or part thereofincluding a transgenic seed, comprises a transgene encoding a Δ8desaturase and/or a Δ9 elongase.

In a further aspect, the present invention provides a method ofproducing an oilseed, the method comprising

i) growing a transgenic oilseed plant according to the invention undersuitable conditions, and

ii) harvesting the seed of the plant.

In a further aspect, the invention provides a part of the transgenicplant of the invention, wherein said part comprises an increased levelof LC-PUFA in its fatty acid relative to the corresponding part from anisogenic non-transformed plant.

Preferably, said plant part is selected from, but not limited to, thegroup consisting of: a seed, leaf, stem, flower, pollen, roots orspecialised storage organ (such as a tuber).

Previously, it has not been shown that LC-PUFA can be produced in plantseeds, nor that these LC-PUFA can be incorporated into plant oils suchas triacylglycerol.

Thus, in another aspect the present invention provides a transgenic seedcomprising a LC-PUFA.

Preferably, the LC-PUFA is selected from the group consisting of:

i) EPA,

ii) DPA,

iii) DHA,

iv) EPA and DPA, and

v) EPA. DHA, and DPA.

More preferably, the LC-PUFA is selected from the group consisting of:

i) DPA,

ii) DHA, or

iii) DHA and DPA.

Even more preferably, the LC-PUFA is EPA, DHA, and DPA.

Preferably, the seed is derived from an isogenic non-transgenic seedwhich produces LA and/or ALA. More preferably, the isogenicnon-transgenic seed comprises a higher concentration of ALA than LA inits fatty acids. Even more preferably, the isogenic non-transgenic seedcomprises at least about 13% ALA or at least about 27% ALA or at leastabout 50% ALA in its fatty acid.

Preferably, the total fatty acid in the oil of the seed comprises atleast 9% C20 fatty acids.

Preferably, the seed is derived from an oilseed plant. More preferably,the oilseed plant is oilseed rape (Brassica napus), maize (Zea mays),sunflower (Helianthus annuus), soybean (Glycine max), sorghum (Sorghumbicolor), flax (Linum usitatissimum), sugar (Saccharum officinarum),beet (Beta vulgaris), cotton (Gossypium hirsutum), peanut (Arachishypogaea), poppy (Papaver somniferum), mustard (Sinapis alba), castorbean (Ricinus communis), sesame (Sesamum indicum), or safflower(Carthamus tinctorius).

It is preferred that the seed has a germination rate which issubstantially the same as that of the isogenic non-transgenic seed.

It is further preferred that the timing of germination of the seed issubstantially the same as that of the isogenic non-transgenic seed.

Preferably, at least 25%, or at least 50%, or at least 75% of theLC-PUFA in the seed form part of triacylglycerols.

Surprisingly, the present inventors have found that transgenic seedsproduced using the methods of the invention have levels of ALA and LAwhich are substantially the same as those of an isogenic non-transgenicseed. As a result, it is preferred that the transgenic seed has levelsof ALA and LA which are substantially the same as those of an isogenicnon-transgenic seed. Furthermore, it was surprising to note that thelevels of monounsaturated fatty acids were decreased in transgenic seedsproduced using the methods of the invention. Accordingly, in a furtherpreferred embodiment, the transgenic seed has decreased levels ofmonounsaturated fatty acids when compared to an isogenic non-transgenicseed.

In another aspect, the present invention provides a method of producinga transgenic seed according to the invention, the method comprising

i) introducing into a progenitor cell of a seed one or morepolynucleotides which encode at least two enzymes each of which is aΔ5/Δ6 bifunctional desaturase, Δ5 desaturase, Δ6 desaturase, Δ5/Δ6bifunctional elongase, Δ5 elongase, Δ6 elongase, Δ4 desaturase, Δ9elongase, or Δ8 desaturase, wherein the one or more polynucleotides areoperably linked to one or more promoters that are capable of directingexpression of said polynucleotides in the cell, thereby producing arecombinant progenitor cell,

ii) culturing said recombinant progenitor cell to produce a plant whichcomprises said transgenic seed, and

iii) recovering the seed from the plant so produced.

In yet a further aspect, the present invention provides a method ofproducing a transgenic seed comprising cultivating a transgenic plantwhich produces the transgenic seed of the invention, and harvesting saidtransgenic seed from the plant.

In a further aspect, the invention provides an extract from thetransgenic plant of the invention, or a plant part of the invention, ora seed of the invention, wherein said extract comprises an increasedlevel of LC-PUFA in its fatty acid relative to a corresponding extractfrom an isogenic non-transformed plant.

Preferably, the extract is substantially purified oil comprising atleast 50% triacylglycerols.

In a further aspect, the present invention provides a non-humantransgenic animal comprising at least one recombinant cell according tothe invention.

Also provided is a method of producing a LC-PUFA, the method comprisingculturing, under suitable conditions, a recombinant cell according tothe invention.

In one embodiment, the cell is of an organism suitable for fermentationand the method further comprises exposing the cell to at least oneLC-PUFA precursor. Preferably, the LC-PUFA precursor is at least one oflinoleic acid or α-linolenic acid. In a particular embodiment, theLC-PUFA precursor is provided in a vegetable oil.

In another embodiment, the cell is an algal cell and the method furthercomprises growing the algal cell under suitable conditions forproduction of said LC-PUFA.

In a further aspect, the present invention provides a method ofproducing one or more LC-PUFA, the method comprising cultivating, undersuitable conditions, a transgenic plant of the invention.

In another aspect, the present invention provides a method of producingoil comprising at least one LC-PUFA, comprising obtaining the transgenicplant of the invention, or the plant part of the invention, or the seedof the invention, and extracting oil from said plant, plant part orseed.

Preferably, said oil is extracted from the seed by crushing said seed.

In another aspect, the present invention provides a method of producingDPA from EPA, the method comprising exposing EPA to a polypeptide of theinvention and a fatty acid precursor, under suitable conditions.

In an embodiment, the method occurs in a cell which uses thepolyketide-like system to produce EPA.

In yet another aspect, the present invention provides a fermentationprocess comprising the steps of:

i) providing a vessel containing a liquid composition comprising a cellof the invention and constituents required for fermentation and fattyacid biosynthesis; and

ii) providing conditions conducive to the fermentation of the liquidcomposition contained in said vessel.

Preferably, a constituent required for fermentation and fatty acidbiosynthesis is LA.

Preferably, the cell is a yeast cell.

In another aspect, the present invention provides a compositioncomprising a cell of the invention, or an extract or portion thereofcomprising LC-PUFA, and a suitable carrier.

In another aspect, the present invention provides a compositioncomprising the transgenic plant of the invention, or the plant part ofthe invention, or the seed of the invention, or an extract or portionthereof comprising LC-PUFA, and a suitable carrier.

In yet another aspect, the present invention provides a feedstuffcomprising a cell of the invention, a plant of the invention, the plantpart of the invention, the seed of the invention, an extract of theinvention, the product of the method of the invention, the product ofthe fermentation process of the invention, or a composition of theinvention.

Preferably, the feedstuff at least comprises DPA, wherein at least oneenzymatic reaction in the production of DPA was performed by arecombinant enzyme in a cell.

Furthermore, it is preferred that the feedstuff comprises at leastcomprises DHA, wherein at least one enzymatic reaction in the productionof DHA was performed by a recombinant enzyme in a cell.

In a further aspect, the present invention provides a method ofpreparing a feedstuff, the method comprising admixing a cell of theinvention, a plant of the invention, the plant part of the invention,the seed of the invention, an extract of the invention, the product ofthe method of the invention, the product of the fermentation process ofthe invention, or a composition of the invention, with a suitablecarrier. Preferably, the feedstuff is for consumption by a mammal or afish.

In a further aspect, the present invention provides a method ofincreasing the levels of a LC-PUFA in an organism, the method comprisingadministering to the organism a cell of the invention, a plant of theinvention, the plant part of the invention, the seed of the invention,an extract of the invention, the product of the method of the invention,the product of the fermentation process of the invention, or acomposition of the invention, or a feedstuff of the invention.

Preferably, the administration route is oral.

Preferably, the organism is a vertebrate. More preferably, thevertebrate is a human, fish, companion animal or livestock animal.

In a further aspect, the present invention provides a method of treatingor preventing a condition which would benefit from a LC-PUFA, the methodcomprising administering to a subject a cell of the invention, a plantof the invention, the plant part of the invention, the seed of theinvention, an extract of the invention, the product of the method of theinvention, the product of the fermentation process of the invention, ora composition of the invention, or a feedstuff of the invention.

Preferably, the condition is arrhythmia's, angioplasty, inflammation,asthma, psoriasis, osteoporosis, kidney stones. AIDS, multiplesclerosis, rheumatoid arthritis, Crohn's disease, schizophrenia, cancer,foetal alcohol syndrome, attention deficient hyperactivity disorder,cystic fibrosis, phenylketonuria, unipolar depression, aggressivehostility, adrenoleukodystophy, coronary heart disease, hypertension,diabetes, obesity. Alzheimer's disease, chronic obstructive pulmonarydisease, ulcerative colitis, restenosis after angioplasty, eczema, highblood pressure, platelet aggregation, gastrointestinal bleeding,endometriosis, premenstrual syndrome, myalgic encephalomyelitis, chronicfatigue after viral infections or ocular disease.

Whilst providing the subject with any amount of LC-PUFA will bebeneficial to the subject, it is preferred that an effective amount totreat the condition is administered.

In another aspect, the present invention provides for the use of a cellof the invention, a plant of the invention, the plant part of theinvention, the seed of the invention, an extract of the invention, theproduct of the method of the invention, the product of the fermentationprocess of the invention, or a composition of the invention, or afeedstuff of the invention, for the manufacture of a medicament fortreating or preventing a condition which would benefit from a LC-PUFA.

The Caenorhabditis elegans Δ6 elongase has previously been expressed inyeast and been shown to convert octadecatetraenoic acid toeicosatetraenoic acid. However, the present inventors have surprisinglyfound that this enzyme also possesses Δ5 elongase activity, being ableto convert eicosapentaenoic acid to docosapentaenoic acid.

In a further aspect, the present invention provides a method ofproducing an unbranched LC-PUFA comprising 22 carbon atoms, the methodcomprising incubating an unbranched 20 carbon atom LC-PUFA with apolypeptide selected from the group consisting of:

i) a polypeptide comprising an amino acid sequence as provided in SEQ IDNO:2 or SEQ ID NO:14,

ii) a polypeptide comprising an amino acid sequence which is at least50% identical to SEQ ID NO:2 or SEQ ID NO:14, and

iii) a biologically active fragment of i) or ii),

wherein the polypeptide also has Δ6 elongase activity.

Preferably, the unbranched LC-PUFA comprising 22 carbon atoms is DPA,and the unbranched 20 carbon atom LC-PUFA is EPA.

Preferably, the method is performed within a recombinant cell whichproduces the polypeptide and EPA.

In yet a further aspect, the present invention provides a substantiallypurified antibody, or fragment thereof, that specifically binds apolypeptide of the invention.

In another aspect, the present invention provides a method ofidentifying a recombinant cell, tissue or organism capable ofsynthesising one or more LC-PUFA, the method comprising detecting thepresence in said cell, tissue or organism of one or more polynucleotideswhich encode at least two enzymes each of which is a Δ5/Δ6 bifunctionaldesaturase, Δ5 desaturase, Δ6 desaturase, Δ5/Δ6 bifunctional elongase,Δ5 elongase, Δ6 elongase, Δ4 desaturase, Δ9 elongase, or Δ8 desaturase,wherein the one or more polynucleotides are operably linked to one ormore promoters that are capable of directing expression of saidpolynucleotides in the cell, tissue or organism.

Preferably, the method comprises a nucleic acid amplification step, anucleic acid hybridisation step, a step of detecting the presence of atransgene in the cell, tissue or organism, or a step of determining thefatty acid content or composition of the cell, tissue or organism,

Preferably, the organism is an animal, plant, angiosperm plant ormicroorganism.

In another aspect, the present invention provides a method of producingDPA from EPA, the method comprising exposing EPA to a Δ5 elongase of theinvention and a fatty acid precursor, under suitable conditions.

Preferably, the method occurs in a cell which uses the polyketide-likesystem to produce EPA.

Naturally, recombinant (transgenic) cells, plants, non-human animalscomprising a new polynucleotide provided herein may also produce otherelongase and/or desaturases such as those defined herein.

As will be apparent, preferred features and characteristics of oneaspect of the invention are applicable to many other aspects of theinvention.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

The invention is hereinafter described by way of the followingnon-limiting Examples and with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG. 1. Possible pathways of ω3 and ω6 LC-PUFA synthesis. The sectorslabelled I, II, III, and IV correspond to the ω6 (Δ6), ω3 (Δ6), ω6 (Δ8),and ω3 (Δ) pathways, respectively. Compounds in sectors I and III are ω6compounds, while those in sectors II and IV are ω3 compounds. “Des”refers to desaturase steps in the pathway catalysed by desaturases asindicated, while “Elo” refers to elongase steps catalysed by elongasesas indicated. The thickened arrow indicates the Δ5 elongase step. Thedashed arrows indicate the steps in the “Sprecher” pathway that operatesin mammalian cells for the production of DHA from DPA.

FIG. 2. Distribution of LC-PUFA in microalgal classes. Chlorophyceae andPrasinophyceae are described as “green algae”, Eustigmatophyceae as“yellow-green algaae”, Rhodophyceae as “red algae”, andBacillariophyceae and Prymnesiophyceae as diatoms and golden brownalgae.

FIG. 3. Genetic construct for expression of LC-PUFA biosynthesis genesin plant cells.

FIG. 4. PILEUP of desaturase enzymes. d8-atg—Pavlova salina Δ8desaturase; euglena—AAD45877 (Δ8 desaturase, Euglena gracilis);rhizopus—AAP83964 (Δ6 desaturase, Rhizopus sp. NK030037); mucor—BAB69055(Δ6 desaturase, Mucor circinelloides); mortierella—AAL73948 (Δ6desaturase, Mortierella isabellina); malpina—BAA85588 (Δ6 desaturase,Mortierella alpina); physcomitrella—CAA11032 (Δ6 acyl-lipid desaturase,Physcomitrella patens); ceratadon—CAB94992 (Δ6 fatty acid acetylenase,Ceratodon purpureus).

FIG. 5. Southern blot of PCR products, hybridized to Elo1 or Elo2probes.

FIG. 6. PILEUP of elongase enzymes.

FIG. 7. Transgene constructs used to express genes encoding LC-PUFAbiosynthetic enzymes in Arabidopsis. The “EPA construct” pSSP-5/6D.6E(also called pZebdesatCeloPWvec8 in Example 5) (FIG. 7A) contained thezebra-fish dual function Δ5/Δ6-desaturase (D5/D6Des) and the nematodeΔ6-elongase (D6Elo) both driven by the truncated napin promoter (Fp1),and the hygromycin resistance selectable marker gene (hph) driven by theCaMV-35S (35SP) promoter. The “DHA construct” pXZP355 (FIG. 7B)comprised the Pavlova salina Δ4-desaturase (D4Des) and Δ5-elongase(D5Elo) genes both driven by the truncated napin promoter (Fp1), and thekanamycin resistance selectable marker gene (nptII) driven by thenopaline synthase promoter (NosP). All genes were flanked at the 3′ endby the nopaline synthase terminator (NosT).

FIG. 8. A. Gas chromatogram (GLC) showing fatty acid profile forArabidopsis thaliana line DO11 carrying EPA and DHA gene constructs. B.Mass spectra for EPA and DHA obtained from Arabidopsis thaliana lineDO11.

FIG. 9. Autoradiograms of dot-blot hybridisations carried out under lowstringency or high stringency conditions, as described in Example 12, toDNA from various microalgal species indicated at the top, usingradiolabelled probes consisting of P. salina LC-PUFA gene coding regionsas indicated on the right.

FIG. 10. Amino acid sequence alignment of Δ6- and Δ8-desaturases fromhigher plants. The amino acid sequences of Δ6-desaturases from E.plantagineum (EplD6Des) (SEQ ID NO:64), E. gentianoides (EgeD6Des,accession number AY055117) (SEQ ID NO:65), E. pitardii (EpiD6Des,AY055118) (SEQ ID NO:66), Borago officinalis (BofD6Des, U79010) (SEQ IDNO:67) and Δ8-desaturases from B. officinalis (BofD8Des, AF133728) (SEQID NO:68), Helianthus annus (HanD8Des, S68358) (SEQ ID NO:69), andArabidopsis thaliana (AtD8DesA. AAC62885.1; and AtD8DesB, CAB71088.1)(SEQ ID NO:70 and SEQ ID NO:71 respectively) were aligned by PILEUP(GCG, Wisconsin, USA). HBI, HBII, HBIII are three conserved histidineboxes. F1 and R1 are the corresponding regions for the degenerateprimers EpD6Des-F1 and EpD6Des-R1 used to amplify the cDNA.Them-terminal cytochrome b₅ domain with conserved HPGG motif is alsoindicated.

FIG. 11. Variant EplD6Des enzymes isolated and representative enzymeactivities. EplD6Des with cytochrome b5, histidine boxes I, II, and IIIare shown as b5, HBI, HBII, HBIII respectively. Variants isolated areshown in panel A in the format: wild-type amino acid—positionnumber—variant amino acid. Empty diamonds indicate mutants withsignificant reduction of enzyme activity, while solid diamonds indicatethe variants with no significant effect on enzyme activity. Panel Bshows the comparison of GLA and SDA production in transgenic tobaccoleaves from two variants with that of wild-type enzyme.

FIG. 12. Alternative pathways for synthesis of the ω3 LC-PUFA SDA(18:4), EPA (20:5) and DHA (22:6) from ALA (18:3). Desaturases,elongases and acyltransferases are shown as solid, open and dashedarrows respectively. Chain elongation occurs only on acyl-CoAsubstrates, whereas desaturation can occur on either acyl-PC [A&B] oracyl-CoA substrates [C]. The acyl-PC or acyl-CoA substrate preference ofthe final Δ4-desaturase step has not yet been determined. Pathwaysinvolving acyl-PC desaturases require acyltransferase-mediated shuttlingof acyl groups between the PC and CoA substrates. Panels A and B showthe “Δ6 pathway” and “Δ8 pathway” variants of the acyl-PC desaturasepathway respectively. Panel C shows the pathway expressed in the currentstudy in which the acyl-CoA Δ6 and Δ5 desaturase activities were encodedby the zebra-fish Δ6/Δ5 dual-function desaturase. Synthesis of ω6LC-PUFA such as ARA (20:4) occurs by the same set of reactions butcommencing with LA (18:2) as the initial substrate.

FIG. 13. Growth rates of Synechococcus 7002 at 22° C., 25° C., 30° C.

FIG. 14. Synechococcus 7002 linoleic and linolenic acid levels atvarious growth temperatures.

KEY TO THE SEQUENCE LISTING

SEQ ID NO:1—Δ8 desaturase from Pavlova salina.

SEQ ID NO:2—Δ5 elongase from Pavlova salina.

SEQ ID NO:3—Δ9 elongase from Pavlova salina.

SEQ ID NO:4—Δ4 desaturase from Pavlova salina.

SEQ ID NO:5—cDNA encoding open reading frame of Δ8 desaturase fromPavlova salina.

SEQ ID NO:6—Full length cDNA encoding of Δ8 desaturase from Pavlovasalina.

SEQ ID NO:7—cDNA encoding open reading frame of Δ5 elongase from Pavlovasalina.

SEQ ID NO:8—Full length cDNA encoding of Δ5 elongase from Pavlovasalina.

SEQ ID NO:9—cDNA encoding open reading frame of Δ9 elongase from Pavlovasalina.

SEQ ID NO:10—Full length cDNA encoding of Δ9 elongase from Pavlovasalina.

SEQ ID NO: 11—Partial cDNA encoding N-terminal portion of Δ4 desaturasefrom Pavlova salina.

SEQ ID NO:12—cDNA encoding open reading frame of Δ4 desaturase fromPavlova salina.

SEQ ID NO:13—Full length cDNA encoding Δ4 desaturase from Pavlovasalina.

SEQ ID NO:14—Δ5/Δ6 bifunctional elongase from Caenorhabditis elegans.

SEQ ID NO:15—Δ5/Δ6 bifunctional desaturase from Danio rerio (zebrafish).

SEQ ID NO: 16—Δ5 desaturase from humans (Genbank Accession No:AAF29378),

SEQ ID NO:17—Δ5 desaturase from Pythium irregulare (Genbank AccessionNo: AAL13311).

SEQ ID NO:18—Δ5 desaturase from Thrausrochytrium sp. (Genbank AccessionNo: AAM09687).

SEQ ID NO:19—Δ5 desaturase from Mortierella alpina (Genbank AccessionNo: O74212).

SEQ ID NO:20—Δ5 desaturase from Caenorhabditis elegans (GenbankAccession No: T43319).

SEQ ID NO:21—Δ6 desaturase from humans (Genbank Accession No: AAD20018).

SEQ ID NO:22—Δ6 desaturase from mouse (Genbank Accession No:NP_(—)062673).

SEQ ID NO:23—Δ6 desaturase from Pythium irregulare (Genbank AccessionNo: AAL13310).

SEQ ID NO:24—Δ6 desaturase from Borago officinalis (Genbank AccessionNo: AAD01410).

SEQ ID NO:25—Δ6 desaturase from Anemone leveillei (Genbank Accession No:AAQ10731),

SEQ ID NO:26—Δ6 desaturase from Ceratodon purpureus (Genbank AccessionNo: CAB94993).

SEQ ID NO:27—Δ6 desaturase from Physcomitrella patens (Genbank AccessionNo: CAA11033).

SEQ ID NO:28—Δ6 desaturase from Mortierella alpina (Genbank AccessionNo: BAC82361).

SEQ ID NO:29—Δ6 desaturase from Caenorhabditis elegans (GenbankAccession No: AAC15586).

SEQ ID NO:30—Δ5 elongase from humans (Genbank Accession No:NP_(—)068586).

SEQ ID NO:31—Δ6 elongase from Physcomitrella patens (Genbank AccessionNo: AAL84174).

SEQ ID NO:32—Δ6 elongase from Mortierella alpina (Genbank Accession No:AAF70417).

SEQ ID NO:33—Δ4 desaturase from Thraustochytrium sp. (Genbank AccessionNo: AAM09688).

SEQ ID NO:34—Δ4 desaturase from Euglena gracilis (Genbank Accession No:AAQ19605).

SEQ ID NO:35—Δ9 elongase from Isochrysis galbana (Genbank Accession No:AAL37626).

SEQ ID NO:36—Δ8 desaturase from Euglena gracilis (Genbank Accession No:AAD45877).

SEQ ID NO:37—cDNA encoding Δ5/Δ6 bifunctional elongase fromCaenorhabditis elegans.

SEQ ID NO:38—cDNA encoding Δ5/Δ6 bifunctional desaturase from Daniorerio (zebrafish).

SEQ ID NO's:39 to 42, 46, 47, 50, 51, 53, 54, 56, 57, 81, 82, 83, 84 and87—Oligonucleotide primers.

SEQ ID NO's:43 to 45, 48, 49 and 52—Conserved motifs of variousdesaturases/elongases.

SEQ ID NO:55—Partial cDNA encoding Pavlova salina FAE-like elongase.

SEQ ID NO:58—Full length cDNA encoding Δ5 desaturase from Pavlovasalina.

SEQ ID NO:59—cDNA encoding open reading frame of Δ5 desaturase fromPavlova salina.

SEQ ID NO:60—Δ5 desaturase from Pavlova salina.

SEQ ID NO's 61 and 62—Fragments of Echium pitardii Δ6 desaturase.

SEQ ID NO:63—cDNA encoding open reading frame of Δ6 desaturase fromEchium plantagineum.

SEQ ID NO:64—Δ6 desaturase from Echium plantagineum.

SEQ ID NO:65—Δ6 desaturase from Echium gentianoides (Genbank AccessionNo: AY055117).

SEQ ID NO:66—Δ6 desaturase from Echium pitardii (Genbank Accession No:AY055118).

SEQ ID NO:67—Δ6 desaturase from Borago officinalis (Genbank AccessionNo: U79010).

SEQ ID NO:68—Δ8 desaturase from Borago officinalis (Genbank AccessionNo: AF133728).

SEQ ID NO:69—Δ8 desaturase from Helianthus annus (Genbank Accession No:S68358).

SEQ ID NO:70—Δ8 desaturaseA from Arabiposis thaliana (Genbank AccessionNo: AAC62885.1).

SEQ ID NO:71—Δ8 desaturaseB from Arabiposis thaliana (Genbank AccessionNo: CAB71088.1).

SEQ ID NO:72 and 73—Conserved motifs of Δ6- and Δ8-desaturases.

SEQ ID NO:74—Δ6 elongase from Thraustochytrium sp. (Genbank AccessionNo: AX951565).

SEQ ID NO:75—Δ9 elongase from Danio rerio (Genbank Accession No:NM_(—)199532).

SEQ ID NO:76—Δ9 elongase from Pavlova lutheri.

SEQ ID NO:77—Δ5 elongase from Danio rerio (Genbank Accession No:AF532782).

SEQ ID NO:78—Δ5 elongase from Pavlova lutheri.

SEQ ID NO:79—Partial gene sequence from Heterocapsa niei encoding anelongase.

SEQ ID NO:80—Protein encoded by SEQ ID NO:79, presence of stop codonsuggests an intron in SEQ ID NO:79.

SEQ ID NO:85—Δ9 elongase from Pavlova salina, encoded by alternate startcodon at position 31 of SEQ ID NO:9.

SEQ ID NO:86—Δ9 elongase from Pavlova salina, encoded by alternate startcodon at position 85 of SEQ ID NO:9.

SEQ ID NO:88—Partial elongase amino acid sequence from Melosira sp.

SEQ ID NO:89—cDNA sequence encoding partial elongase from Melosira sp.

DETAILED DESCRIPTION OF THE INVENTION

General Techniques and Definitions

Unless specifically defined otherwise, all technical and scientificterms used herein shall be taken to have the same meaning as commonlyunderstood by one of ordinary skill in the art (e.g., in cell culture,plant biology, molecular genetics, immunology, immunohistochemistry,protein chemistry, fatty acid synthesis, and biochemistry).

Unless otherwise indicated, the recombinant nucleic acid, recombinantprotein, cell culture, and immunological techniques utilized in thepresent invention are standard procedures, well known to those skilledin the art. Such techniques are described and explained throughout theliterature in sources such as, J. Perbal, A Practical Guide to MolecularCloning, John Wiley and Sons (1984), J. Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press(1989), T. A. Brown (editor), Essential Molecular Biology: A PracticalApproach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D.Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRLPress (1995 and 1996), and F. M. Ausubel et al. (editors), CurrentProtocols in Molecular Biology, Greene Pub. Associates andWiley-Interscience (1988, including all updates until present), EdHarlow and David Lane (editors) Antibodies: A Laboratory Manual, ColdSpring Harbour Laboratory, (1988), and J. E. Coligan et al. (editors)Current Protocols in Immunology, John Wiley & Sons (including allupdates until present), and are incorporated herein by reference.

As used herein, the terms “long-chain polyunsaturated fatty acid”,“LC-PUFA” or “C20+ polyunsaturated fatty acid” refer to a fatty acidwhich comprises at least 20 carbon atoms in its carbon chain and atleast three carbon-carbon double bonds. As used herein, the term “verylong-chain polyunsaturated fatty acid”, “VLC-PUFA” or “C22+polyunsaturated fatty acid” refers to a fatty acid which comprises atleast 22 carbon atoms in its carbon chain and at least threecarbon-carbon double bonds. Ordinarily, the number of carbon atoms inthe carbon chain of the fatty acids refers to an unbranched carbonchain. If the carbon chain is branched, the number of carbon atomsexcludes those in sidegroups. In one embodiment, the long-chainpolyunsaturated fatty acid is an ω3 fatty acid, that is, having adesaturation (carbon-carbon double bond) in the third carbon-carbon bondfrom the methyl end of the fatty acid. In another embodiment, thelong-chain polyunsaturated fatty acid is an ω6 fatty acid, that is,having a desaturation (carbon-carbon double bond) in the sixthcarbon-carbon bond from the methyl end of the fatty acid. In a furtherembodiment, the long-chain polyunsaturated fatty acid is selected fromthe group consisting of; arachidonic acid (ARA, 20:4Δ5,8,11,14; ω6),eicosatetraenoic acid (ETA, 20:4Δ8,11,14,17, ω3) eicosapentaenoic acid(EPA, 20:5Δ5,8,11,14,17; ω3), docosapentaenoic acid (DPA,22:5Δ7,10,13,16,19, ω3), or docosahexaenoic acid (DHA,22:6Δ4,7,10,13,16,19, ω3). The LC-PUFA may also be dihomo-γ-linoleicacid (DGLA) or eicosatrienoic acid (ETrA, 20:3Δ11,14,17, ω3). It wouldreadily be apparent that the LC-PUFA that is produced according to theinvention may be a mixture of any or all of the above and may includeother LC-PUFA or derivatives of any of these LC-PUFA. In a preferredembodiment, the ω3 fatty acid is EPA, DPA, or DHA, or even morepreferably DPA or DHA.

Furthermore, as used herein the terms “long-chain polyunsaturated fattyacid” or “very long-chain polyunsaturated fatty acid” refer to the fattyacid being in a free state (non-esterified) or in an esterified formsuch as part of a triglyceride, diacylglyceride, monoacylglyceride,acyl-CoA bound or other bound form. The fatty acid may be esterified asa phospholipid such as a phosphatidylcholine, phosphatidylethanolamine,phosphatidylserine, phosphatidylglycerol, phosphatidylinositol ordiphosphatidylglycerol forms. Thus, the LC-PUFA may be present as amixture of forms in the lipid of a cell or a purified oil or lipidextracted from cells, tissues or organisms. In preferred embodiments,the invention provides oil comprising at least 75% or 85%triacylglycerols, with the remainder present as other forms of lipidsuch as those mentioned, with at least said triacylglycerols comprisingthe LC-PUFA. The oil may be further purified or treated, for example byhydrolysis with a strong base to release the free fatty acid, or byfractionation, distillation or the like.

As used herein, the abbreviations “LC-PUFA” and “VLC-PUFA” can refer toa single type of fatty acid, or to multiple types of fatty acids. Forexample, a transgenic plant of the invention which produces LC-PUFA mayproduce EPA, DPA and DHA.

The desaturase and elongase proteins and genes encoding them that may beused in the invention are any of those known in the art or homologues orderivatives thereof. Examples of such genes and the encoded proteinsizes are listed in Table 1. The desaturase enzymes that have been shownto participate in LC-PUFA biosynthesis all belong to the group ofso-called “front-end” desaturases which are characterised by thepresence of a cytochrome b₅-like domain at the N-terminus of eachprotein. The

TABLE 1 Cloned genes involved in LC-PUFA biosynthesis. Protein Type ofAccession size Enzyme organism Species Nos. (aa's) References Δ4- AlgaeEuglena gracilis AY278558 541 Meyer et al., desaturase 2003 Pavlovalutherii AY332747 445 Tonon et al., 2003 Thraustochytrium AF489589 519Qiu et al., 2001 sp. Thraustochytrium AF391543-5 515 (NCBI) aureum Δ5-Mammals Homo sapiens AF199596 444 Cho et al., 1999b desaturase Leonardet al., 2000b Nematode Caenorhabditis AF11440, 447 Michaelson et elegansNM_069350 al., 1998b; Watts and Browse, 1999b Fungi Mortierella AF067654446 Michaelson et alpina al., 1998a; Knutzon et al., 1998 PythiumAF419297 456 Hong et al., irregulare 2002a Dictyostelium AB022097 467Saito et al., 2000 discoideum Saprolegnia 470 WO02081668 diclina DiatomPhaeodactylum AY082392 469 Domergue et al., tricornutum 2002 AlgaeThraustochytrium AF489588 439 Qiu et al., 2001 sp Thraustochytrium 439WO02081668 aureum Isochrysis 442 WO02081668 galbana Moss MarchantiaAY583465 484 Kajikawa et al., polymorpha 2004 Δ6- Mammals Homo sapiensNM_013402 444 Cho et al., 1999a; desaturase Leonard et al., 2000 Musmusculus NM_019699 444 Cho et al., 1999a Nematode Caenorhabditis Z70271443 Napier et al., 1998 elegans Plants Borago officinales U79010 448Sayanova et al., 1997 Echium AY055117 Garcia-Maroto et AY055118 al.,2002 Primula vialii AY234127 453 Sayanova et al., 2003 Anemone leveilleiAF536525 446 Whitney et al., 2003 Mosses Ceratodon AJ250735 520 Sperlinget al., 2000 purpureus Marchantia AY583463 481 Kajikawa et al.,polymorpha 2004 Physcomitrella Girke et al., 1998 patens FungiMortierella alpina AF110510 457 Huang et al., 1999; AB020032 Sakuradaniet al., 1999 Pythium AF419296 459 Hong et al., 2002a irregulare MucorAB052086 467 NCBI* circinelloides Rhizopus sp. AY320288 458 Zhang etal., 2004 Saprolegnia 453 WO02081668 diclina Diatom PhaeodactylumAY082393 477 Domergue et al., tricornutum 2002 Bacteria SynechocystisL11421 359 Reddy et al., 1993 Algae Thraustochytrium 456 WO02081668aureum Bifunctional Fish Danio rerio AF309556 444 Hastings et al., Δ5/Δ62001 desaturase C20 Δ8- Algae Euglena gracilis AF139720 419 Wallis andBrowse, desaturase 1999 Plants Borago officinales AF133728 Δ6-elongaseNematode Caenorhabditis NM_069288 288 Beaudoin et al., elegans 2000Mosses Physcomitrella AF428243 290 Zank et al., 2002 patens MarchantiaAY583464 290 Kajikawa et al., polymorpha 2004 Fungi Mortierella AF206662318 Parker-Barnes et alpina al., 2000 Algae Pavlova lutheri** 501 WO03078639 Thraustochytrium AX951565 271 WO 03093482 ThraustochytriumAX214454 271 WO 0159128 sp** PUFA- Mammals Homo sapiens AF231981 299Leonard et al., elongase 2000b; Leonard et al., 2002 Rattus norvegicusAB071985 299 Inagaki et al., 2002 Rattus AB071986 267 Inagaki et al.,norvegicus** 2002 Mus musculus AF170907 279 Tvrdik et al., 2000 Musmusculus AF170908 292 Tvrdik et al., 2000 Fish Danio rerio AF532782 291Agaba et al., 2004 (282) Danio rerio** NM_199532 266 Lo et al., 2003Worm Caenorhabditis Z68749 309 Abbott et al 1998 elegans Beaudoin et al2000 Algae Thraustochytrium AX464802 272 WO 0208401-A2 aureum** Pavlovalutheri** ? WO 03078639 Δ9-elongase Algae Isochrysis AF390174 263 Qi etal., 2002 galbana *http://www.ncbi.nlm.nih.gov/ **Function notproven/not demonstratedcytochrome b₅-like domain presumably acts as a receptor of electronsrequired for desaturation (Napier et al., 1999; Sperling and Heinz,2001).

Activity of any of the elongases or desaturases for use in the inventionmay be tested by expressing a gene encoding the enzyme in a cell suchas, for example, a yeast cell or a plant cell, and determining whetherthe cell has an increased capacity to produce LC-PUFA compared to acomparable cell in which the enzyme is not expressed.

Unless stated to the contrary, embodiments of the present inventionwhich relate to cells, plants, seeds, etc, and methods for theproduction thereof, and that refer to at least “two enzymes” (or atleast “three enzymes” etc) of the list that is provided means that thepolynucleotides encode at least two “different” enzymes from the listprovided and not two identical (or very similar with only a fewdifferences as to not substantially alter the activity of the encodedenzyme) open reading frames encoding essentially the same enzyme.

As used herein, unless stated to the contrary, the term “substantiallythe same”, or variations thereof, means that two samples being analysed,for example two seeds from different sources, are substantially the sameif they only vary about +/−10% in the trait being investigated.

As used herein, the term “an enzyme which preferentially converts an ω6LC-PUFA into an ω3 LC-PUFA” means that the enzyme is more efficient atperforming said conversion than it is at performing a desaturationreaction outlined in pathways II or III of FIG. 1.

Whilst certain enzymes are specifically described herein as“bifunctional”, the absence of such a term does not necessarily implythat a particular enzyme does not possess an activity other than thatspecifically defined.

Desaturases

As used herein, a “Δ5/Δ6 bifunctional desaturase” or “Δ5/Δ6 desaturase”is at least capable of i) converting α-linolenic acid tooctadecatetraenoic acid, and ii) converting eicosatetraenoic acid toeicosapentaenoic acid. That is, a Δ5/Δ6 bifunctional desaturase is botha Δ5 desaturase and a Δ6 desaturase, and Δ5/Δ6 bifunctional desaturasesmay be considered a sub-class of each of these. A gene encoding abifunctional Δ5-/Δ6-desaturase has been identified from zebrafish(Hasting et al., 2001). The gene encoding this enzyme might represent anancestral form of the “front-end desaturase” which later duplicated andthe copies evolved distinct Δ5- and Δ6-desaturase functions. In oneembodiment, the Δ5/Δ6 bifunctional desaturase is naturally produced by afreshwater species of fish. In a particular embodiment, the Δ5/Δ6bifunctional desaturase comprises

i) an amino acid sequence as provided in SEQ ID NO:15,

ii) an amino acid sequence which is at least 50% identical to SEQ IDNO:15, or

iii) a biologically active fragment of i) or ii).

As used herein, a “Δ5 desaturase” is at least capable of convertingeicosatetraenoic acid to eicosapentaenoic acid. In one embodiment, theenzyme Δ5 desaturase catalyses the desaturation of C20 LC-PUFA,converting DGLA to arachidonic acid (ARA, 20:4ω6) and ETA to EPA(20:5ω3). Genes encoding this enzyme have been isolated from a number oforganisms, including algae (Thraustochytrium sp. Qiu et al., 2001),fungi (M. alpine, Pythium irregulare. P. tricornutum, Dictyostelium),Caenorhabditis elegans and mammals (Table 1). In another embodiment, theΔ5 desaturase comprises (i) an amino acid sequence as provided in SEQ IDNO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 or SEQ IDNO:60, (ii) an amino acid sequence which is at least 50% identical toany one of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQID NO:20 or SEQ ID NO:60, or (iii) a biologically active fragment of i)or ii). In a further embodiment, the Δ5 desaturase comprises (i) anamino acid sequence as provided in SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:20 or SEQ ID NO:60, (ii) an amino acid sequence whichis at least 90% identical to any one of SEQ ID NO:16, SEQ ID NO:17, SEQID NO:18, SEQ ID NO:20 or SEQ ID NO:60, or (iii) a biologically activefragment of i) or ii). In a further embodiment, the Δ5 desaturase isencoded by the protein coding region of one of the Δ5 desaturase geneslisted in Table 1 or gene substantially identical thereto.

As used herein, a “Δ6 desaturase” is at least capable of convertingα-linolenic acid to octadecatetraenoic acid. In one embodiment, theenzyme Δ6 desaturase catalyses the desaturation of C18 LC-PUFA,converting LA to GLA and ALA to SDA. In another embodiment, the Δ6desaturase comprises (i) an amino acid sequence as provided in SEQ IDNO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ IDNO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:64 SEQ IDNO:65, SEQ ID NO:66 or SEQ ID NO:67, (ii) an amino acid sequence whichis at least 50% identical to any one of SEQ ID NO:21, SEQ ID NO:22, SEQID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ IDNO:28, SEQ ID NO:29, SEQ ID NO:64 SEQ ID NO:65, SEQ ID NO:66 or SEQ IDNO:67, or (iii) a biologically active fragment of i) or ii). In afurther embodiment, the Δ6 desaturase comprises an amino acid sequencewhich is at least 90% identical to any one of SEQ ID NO:21, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:64 SEQ ID NO:65, SEQ IDNO:66 or SEQ ID NO:67. In a further embodiment, the Δ6 desaturase isencoded by the protein coding region of one of the Δ6 desaturase geneslisted in Table 1 or gene substantially identical thereto

As used herein, a “Δ4 desaturase” is at least capable of convertingdocosapentaenoic acid to docosahexaenoic acid. The desaturation step toproduce DHA from DPA is catalysed by a Δ4 desaturase in organisms otherthan mammals, and a gene encoding this enzyme has been isolated from thefreshwater protist species Euglena gracilis and the marine speciesThraustochyrrium sp. (Qiu et al., 2001; Meyer et al., 2003). In oneembodiment, the Δ4 desaturase comprises (i) an amino acid sequence asprovided in SEQ ID NO:4, SEQ ID NO:33 or SEQ ID NO:34, (ii) an aminoacid sequence which is at least 50% identical to SEQ ID NO:4, SEQ IDNO:33 or SEQ ID NO:34, or (iii) a biologically active fragment of i) orii). In a further embodiment, the Δ4 desaturase is encoded by theprotein coding region of one of the Δ4 desaturase genes listed in Table1 or gene substantially identical thereto.

As used herein, a “Δ8 desaturase” is at least capable of converting20:3^(Δ11,14,17)ω3 to eicosatetraenoic acid. In one embodiment, the Δ8desaturase is relatively specific for Δ8 substrates. That is, it hasgreater activity in desaturating Δ8 substrates than other substrates, inparticular Δ6 desaturated substrates. In a preferred embodiment, the Δ8desaturase has little or no Δ6 desaturase activity when expressed inyeast cells. In another embodiment, the Δ8 desaturase comprises (i) anamino acid sequence as provided in SEQ ID NO:1, SEQ ID NO:36, SEQ IDNO:68, SEQ ID NO:69, SEQ ID NO:70 or SEQ ID NO:71, (ii) an amino acidsequence which is at least 50% identical to SEQ ID NO:1, SEQ ID NO:36,SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70 or SEQ ID NO:71, or (iii) abiologically active fragment of i) or ii). In further embodiment, the Δ8desaturase comprises (i) an amino acid sequence as provided in SEQ IDNO:1, (ii) an amino acid sequence which is at least 90% identical to SEQID NO:1, or (iii) a biologically active fragment of i) or ii).

As used herein, an “ω3 desaturase” is at least capable of converting LAto ALA and/or GLA to SDA and/or ARA to EPA. Examples of ω3 desaturaseinclude those described by Pereira et al. (2004), Horiguchi et al.(1998), Berberich et al. (1998) and Spychalla et al. (1997). In oneembodiment, a cell of the invention is a plant cell which lacks ω3desaturase activity. Such cells can be produced using gene knockouttechnology well known in the art. These cells can be used tospecifically produce large quantities of ω6 LC-PUFA such as DGLA.

Elongases

Biochemical evidence suggests that the fatty acid elongation consists of4 steps: condensation, reduction, dehydration and a second reduction. Inthe context of this invention, an “elongase” refers to the polypeptidethat catalyses the condensing step in the presence of the other membersof the elongation complex, under suitable physiological conditions. Ithas been shown that heterologous or homologous expression in a cell ofonly the condensing component (“elongase”) of the elongation proteincomplex is required for the elongation of the respective acyl chain.Thus the introduced elongase is able to successfully recruit thereduction and dehydration activities from the transgenic host to carryout successful acyl elongations. The specificity of the elongationreaction with respect to chain length and the degree of desaturation offatty acid substrates is thought to reside in the condensing component.This component is also thought to be rate limiting in the elongationreaction.

Two groups of condensing enzymes have been identified so far. The firstare involved in the extension of saturated and monounsaturated fattyacids (C18-22) such as, for example, the FAE1 gene of Arabidopsis. Anexample of a product formed is erucic acid (22:1) in Brassicas. Thisgroup are designated the FAE-like enzymes and do not appear to have arole in LC-PUFA biosynthesis. The other identified class of fatty acidelongases, designated the ELO family of elongases, are named after theELO genes whose activities are required for the synthesis of the verylong-chain fatty acids of sphingolipids in yeast. Apparent paralogs ofthe ELO-type elongases isolated from LC-PUFA synthesizing organisms likealgae, mosses, fungi and nematodes have been shown to be involved in theelongation and synthesis of LC-PUFA. Several genes encoding suchPUFA-elongation enzymes have also been isolated (Table 1). Such genesare unrelated in nucleotide or amino acid sequence to the FAE-likeelongase genes present in higher plants.

As used herein, a “Δ5/Δ6 bifunctional elongase” or “Δ5/Δ6 elongase” isat least capable of i) converting octadecatetraenoic acid toeicosatetraenoic acid, and ii) converting eicosapentaenoic acid todocosapentaenoic acid. That is, a Δ5/Δ6 bifunctional elongase is both aΔ5 elongase and a Δ6 elongase, and Δ5/Δ6 bifunctional elongases may beconsidered a sub-class of each of these. In one embodiment, the Δ5/Δ6bifunctional elongase is able to catalyse the elongation of EPA to formDPA in a plant cell such as, for example, a higher plant cell, when thatcell is provided with a source of EPA. The EPA may be providedexogenously or preferably endogenously. A gene encoding such an elongasehas been isolated from an invertebrate. C. elegans (Beaudoin et al.,2000) although it was not previously known to catalyse the Δ5-elongationstep. In one embodiment, the Δ5/Δ6 bifunctional elongase comprises (i)an amino acid sequence as provided in SEQ ID NO:2 or SEQ ID NO:14, (ii)an amino acid sequence which is at least 50% identical to SEQ ID NO:2 orSEQ ID NO:14, or (iii) a biologically active fragment of i) or ii).

As used herein, a “Δ5 elongase” is at least capable of convertingeicosapentaenoic acid to docosapentaenoic acid. In one embodiment, theΔ5 elongase is from a non-vertebrate source such as, for example, analgal or fungal source. Such elongases can have advantages in terms ofthe specificity of the elongation reactions carried out (for example theΔ5 elongase provided as SEQ ID NO:2). In a preferred embodiment, the Δ5elongase is relatively specific for C20 substrates over C22 substrates.For example, it may have at least 10-fold lower activity toward C22substrates (elongated to C24 fatty acids) relative to the activitytoward a corresponding C20 substrate when expressed in yeast cells. Itis preferred that the activity when using C20 Δ5 desaturated substratesis high, such as for example, providing an efficiency for the conversionof 20:5ω3 into 22:5ω3 of at least 7% when expressed in yeast cells. Inanother embodiment, the Δ5 elongase is relatively specific for Δ5desaturated substrates over Δ6 desaturated substrates. For example, itmay have at least 10-fold lower activity toward Δ6 desaturated C18substrates relative to Δ5 desaturated C20 substrates when expressed inyeast cells. In a further embodiment, the Δ5 elongase comprises (i) anamino acid sequence as provided in SEQ ID NO:2, SEQ ID NO:30, SEQ IDNO:77 or SEQ ID NO:78, (ii) an amino acid sequence which is at least 50%identical to SEQ ID NO:2, SEQ ID NO:30, SEQ ID NO:77 or SEQ ID NO:78, or(iii) a biologically active fragment of i) or ii). In anotherembodiment, the Δ5 elongase comprises (i) an amino acid sequence asprovided in SEQ ID NO:2, (ii) an amino acid sequence which is at least90% identical to SEQ ID NO:2, or (iii) a biologically active fragment ofi) or ii). In a further embodiment, the Δ5 elongase is encoded by theprotein coding region of one of the Δ5 elongase genes listed in Table 1or gene substantially identical thereto.

As used herein, a “Δ6 elongase” is at least capable of convertingoctadecatetraenoic acid to eicosatetraenoic acid. In one embodiment, theΔ6 elongase comprises (i) an amino acid sequence as provided in SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:74, SEQ IDNO:85, SEQ ID NO:86 or SEQ ID NO:88, (ii) an amino acid sequence whichis at least 50% identical to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:31, SEQID NO:32, SEQ ID NO:74, SEQ ID NO:85, SEQ ID NO:86 or SEQ ID NO:88, or(iii) a biologically active fragment of i) or ii). In anotherembodiment, the Δ6 elongase comprises (i) an amino acid sequence asprovided in SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:32, SEQ ID NO:85, SEQID NO:86 or SEQ ID NO:88, (ii) an amino acid sequence which is at least90% identical to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:32, SEQ ID NO:85,SEQ ID NO:86 or SEQ ID NO:88, or (iii) a biologically active fragment ofi) or ii). In a further embodiment, the Δ6 elongase is encoded by theprotein coding region of one of the Δ6 elongase genes listed in Table 1or gene substantially identical thereto.

In some protist species, LC-PUFA are synthesized by elongation oflinoleic or α-linolenic acid with a C2 unit, before desaturation with Δ8desaturase (FIG. 1 part IV; “Δ8-desaturation” pathway). Δ6 desaturaseand Δ6 elongase activities were not detected in these species. Instead,a Δ9-elongase activity would be expected in such organisms, and insupport of this, a C18 Δ9-elongase gene has recently been isolated fromIsochrysis galbana (Qi et al., 2002). As used herein, a “Δ9 elongase” isat least capable of converting α-linolenic acid to 20:3^(Δ11,14,17)ω3.In one embodiment, the Δ9 elongase comprises (i) an amino acid sequenceas provided in SEQ ID NO:3, SEQ ID NO:35, SEQ ID NO:75, SEQ ID NO:76.SEQ ID NO:85 or SEQ ID NO:86, (ii) an amino acid sequence which is atleast 50% identical to SEQ ID NO:3, SEQ ID NO:35, SEQ ID NO:75, SEQ IDNO:76, SEQ ID NO:85 or SEQ ID NO:86, or (iii) a biologically activefragment of i) or ii). In another embodiment, the Δ9 elongase comprises(i) an amino acid sequence as provided in SEQ ID NO:3, SEQ ID NO:85 orSEQ ID NO:86, (ii) an amino acid sequence which is at least 90%identical to SEQ ID NO:3, SEQ ID NO:85 or SEQ ID NO:86, or (iii) abiologically active fragment of i) or ii). In a further embodiment, theΔ9 elongase is encoded by the protein coding region of the Δ9 elongasegene listed in Table 1 or gene substantially identical thereto. Inanother embodiment, the Δ9 elongase also has Δ6 elongase activity. Theelongase in this embodiment is able to convert SDA to ETA and/or GLA toDGLA (Δ6 elongase activity) in addition to converting ALA to ETrA (Δ9elongase). In a preferred embodiment, such an elongase is from an algalor fungal source such as, for example, the genus Pavlova.

As used herein, a “Δ4 elongase” is at least capable of convertingdocosahexaenoic acid to 24:6^(Δ6,9,12,15,18,21)ω3.

Cells

Suitable cells of the invention include any cell that can be transformedwith a polynucleotide encoding a polypeptide/enzyme described herein,and which is thereby capable of being used for producing LC-PUFA. Hostcells into which the polynucleotide(s) are introduced can be eitheruntransformed cells or cells that are already transformed with at leastone nucleic acid molecule. Such nucleic acid molecule may related toLC-PUFA synthesis, or unrelated. Host cells of the present inventioneither can be endogenously (i.e., naturally) capable of producingproteins of the present invention or can be capable of producing suchproteins only after being transformed with at least one nucleic acidmolecule.

As used herein, the term “cell with an enhanced capacity to synthesize along chain polyunsaturated fatty acid” is a relative term where therecombinant cell of the invention is compared to the native cell, withthe recombinant cell producing more long chain polyunsaturated fattyacids, or a greater concentration of LC-PUFA such as EPA, DPA or DHA(relative to other fatty acids), than the native cell.

The cells may be prokaryotic or eukaryotic. Host cells of the presentinvention can be any cell capable of producing at least one proteindescribed herein, and include bacterial, fungal (including yeast),parasite, arthropod, animal and plant cells. Preferred host cells areyeast and plant cells. In a preferred embodiment, the plant cells areseed cells.

In one embodiment, the cell is an animal cell or an algal cell. Theanimal cell may be of any type of animal such as, for example, anon-human animal cell, a non-human vertebrate cell, a non-humanmammalian cell, or cells of aquatic animals such as fish or crustacea,invertebrates, insects, etc.

An example of a bacterial cell useful as a host cell of the presentinvention is Synechococcus spp. (also known as Synechocystis spp.), forexample Synechococcus elongatus.

The cells may be of an organism suitable for fermentation. As usedherein, the term the “fermentation process” refers to any fermentationprocess or any process comprising a fermentation step. A fermentationprocess includes, without limitation, fermentation processes used toproduce alcohols (e.g., ethanol, methanol, butanol); organic acids(e.g., citric acid, acetic acid, itaconic acid, lactic acid, gluconicacid); ketones (e.g., acetone); amino acids (e.g., glutamic acid); gases(e.g., H₂ and CO₂); antibiotics (e.g., penicillin and tetracycline);enzymes; vitamins (e.g., riboflavin, beta-carotene); and hormones.Fermentation processes also include fermentation processes used in theconsumable alcohol industry (e.g., beer and wine), dairy industry (e.g.,fermented dairy products), leather industry and tobacco industry.Preferred fermentation processes include alcohol fermentation processes,as are well known in the art. Preferred fermentation processes areanaerobic fermentation processes, as are well known in the art.

Suitable fermenting cells, typically microorganisms are able to ferment,i.e., convert, sugars, such as glucose or maltose, directly orindirectly into the desired fermentation product. Examples of fermentingmicroorganisms include fungal organisms, such as yeast. As used herein,“yeast” includes Saccharomyces spp., Saccharomyces cerevisiae,Saccharomyces carlbergensis, Candida spp., Kluveromyces spp., Pichiaspp., Hansenula spp., Trichoderma spp., Lipomyces starkey, and Yarrowialipolytica. Preferred yeast include strains of the Saccharomyces spp.,and in particular, Saccharomyces cerevisiae. Commercially availableyeast include, e.g., Red Star/Lesaffre Ethanol Red (available from RedStar/Lesaffre, USA) FALI (available from Fleischmann's Yeast, a divisionof Burns Philp Food Inc., USA), SUPERSTART (available from Alltech),GERT STRAND (available from Gert Strand AB, Sweden) and FERMIOL(available from DSM Specialties).

Evidence to date suggests that some desaturases expressed heterologouslyin yeast have relatively low activity in combination with someelongases. However, the present inventors have identified that this maybe alleviated by providing a desaturase with the capacity of to use anacyl-CoA form of the fatty acid as a substrate in LC-PUFA synthesis, andthis is thought to be advantageous in recombinant cells other than yeastas well. In this regard, it has also been determined that desaturases ofvertebrate origin are particularly useful for the production of LC-PUFA.Thus in embodiments of the invention, either (i) at least one of theenzymes is a Δ5 elongase that catalyses the conversion of EPA to DPA inthe cell, (ii) at least one of the desaturases is able to act on anacyl-CoA substrate, (iii) at least one desaturase is from vertebrate oris a variant thereof, or (iv) a combination of ii) and iii).

In a particularly preferred embodiment, the host cell is a plant cell,such as those described in further detail herein.

As used herein, a “progenitor cell of a seed” is a cell that dividesand/or differentiates into a cell of a transgenic seed of the invention,and/or a cell that divides and/or differentiates into a transgenic plantthat produces a transgenic seed of the invention.

Levels of LC-PUFA Produced

The levels of the LC-PUFA that are produced in the recombinant cell areof importance. The levels may be expressed as a composition (in percent)of the total fatty acid that is a particular LC-PUFA or group of relatedLC-PUFA, for example the ω3 LC-PUFA or the ω6 LC-PUFA, or the C22+ PUFA,or other which may be determined by methods known in the art. The levelmay also be expressed as a LC-PUFA content, such as for example thepercentage of LC-PUFA in the dry weight of material comprising therecombinant cells, for example the percentage of the dry weight of seedthat is LC-PUFA. It will be appreciated that the LC-PUFA that isproduced in an oilseed may be considerably higher in terms of LC-PUFAcontent than in a vegetable or a grain that is not grown for oilproduction, yet both may have similar LC-PUFA compositions, and both maybe used as sources of LC-PUFA for human or animal consumption.

The levels of LC-PUFA may be determined by any of the methods known inthe art. For example, total lipid may be extracted from the cells,tissues or organisms and the fatty acid converted to methyl estersbefore analysis by gas chromatography (GC). Such techniques aredescribed in Example 1. The peak position in the chromatogram may beused to identify each particular fatty acid, and the area under eachpeak integrated to determine the amount. As used herein, unless statedto the contrary, the percentage of particular fatty acid in a sample isdetermined as the area under the peak for that fatty acid as apercentage of the total area for fatty acids in the chromatogram. Thiscorresponds essentially to a weight percentage (w/w). The identity offatty acids may be confirmed by GC-MS, as described in Example 1.

In certain embodiments, where the recombinant cell is useful in afermentation process such as, for example, a yeast cell, the level ofEPA that is produced may be at least 0.21% of the total fatty acid inthe cell, preferably at least 0.82% or at least 2% and even morepreferably at least 5%.

In other embodiments, the total fatty acid of the recombinant cell maycomprise at least 1.5% EPA, preferably at least 2.1% EPA, and morepreferably at least 2.5%, at least 3.1%, at least 4% or at least 5.1%EPA.

In further embodiments, where the recombinant cell is useful in afermentation process or is a plant cell and DPA is produced, the totalfatty acid in the cell may comprise at least 0.1% DPA, preferably atleast 0.13% or at least 0.15% and more preferably at least 0.5% or atleast 1% DPA.

In further embodiments, the total fatty acid of the cell may comprise atleast 2% C20 LC-PUFA, preferably at least 3% or at least 4% C20 LC-PUFA,more preferably at least 4.7% or at least 7.9% C20 LC-PUFA and mostpreferably at least 10.2% C20 LC-PUFA.

In further embodiments, the total fatty acid of the cell may comprise atleast 2.5% C20 ω3 LC-PUFA, preferably at least 4.1% or more preferablyat least 5% C20 ω3 LC-PUFA.

In other embodiments, where both EPA and DPA are synthesized in a cell,the level of EPA reached is at least 1.5%, at least 2.1% or at least2.5% and the level of DPA at least 0.13%, at least 0.5% or at least1.0%.

In each of these embodiments, the recombinant cell may be a cell of anorganism that is suitable for fermentation such as, for example, aunicellular microorganism which may be a prokaryote or a eukaryote suchas yeast, or a plant cell. In a preferred embodiment, the cell is a cellof an angiosperm (higher plant). In a further preferred embodiment, thecell is a cell in a seed such as, for example, an oilseed or a grain orcereal.

The level of production of LC-PUFA in the recombinant cell may also beexpressed as a conversion ratio, i.e. the amount of the LC-PUFA formedas a percentage of one or more substrate PUFA or LC-PUFA. With regard toEPA, for example, this may be expressed as the ratio of the level of EPA(as a percentage in the total fatty acid) to the level of a substratefatty acid (ALA, SDA, ETA or ETrA). In a preferred embodiment, theconversion efficiency is for ALA to EPA. In particular embodiments, theconversion ratio for production of EPA in a recombinant cell may be atleast 0.5%, at least 1%, or at least 2%. In another embodiment, theconversion efficiency for ALA to EPA is at least 14.6%. In furtherembodiments, the conversion ratio for production of DPA from EPA in arecombinant cell is at least 5%, at least 7%, or at least 10%. In otherembodiments, the total ω3 fatty acids produced that are products of Δ6desaturation (i.e. downstream of 18:3ω3 (ALA), calculated as the sum ofthe percentages for 18:4ω3 (SDA), 20:4ω3 (ETA), 20:5ω3 (EPA) and 22:5ω3(DPA)) is at least 4.2%. In a particular embodiment, the conversionefficiency of ALA to ω3 products through a Δ6 desaturation step and/oran Δ9 elongation step in a recombinant cell, preferably a plant cell,more preferably a seed cell, is at least 22% or at least 24%. Statedotherwise, in this embodiment the ratio of products derived from ALA toALA (products:ALA) in the cell is at least 1:3.6.

The content of the LC-PUFA in the recombinant cell may be maximized ifthe parental cell used for introduction of the genes is chosen such thatthe level of fatty acid substrate that is produced or providedexogenously is optimal. In particular embodiments, the cell produces ALAendogenously at levels of at least 30%, at least 50%, or at least 66% ofthe total fatty acid. The level of LC-PUFA may also be maximized bygrowing or incubating the cells under optimal conditions, for example ata slightly lower temperature than the standard temperature for thatcell, which is thought to favour accumulation of polyunsaturated fattyacid.

There are advantages to maximizing production of a desired LC-PUFA whileminimizing the extent of side-reactions. In a particular embodiment,there is little or no ETrA detected (less than 0.1%) while the level ofEPA is at least 2.1%.

Turning to transgenic plants of the invention, in one embodiment, atleast one plant part synthesizes EPA, wherein the total fatty acid ofthe plant part comprises at least 1.5%, at least 2.1%, or at least 2.5%EPA.

In another embodiment, at least one plant part synthesizes DPA, whereinthe total fatty acid of the plant part comprises at least 0.1%, at least0.13%, or at least 0.5% DPA.

In a further embodiment, at least one plant part synthesizes DHA.

In another embodiment, at least one plant part synthesizes DHA, whereinthe total fatty acid of the plant part comprises at least 0.1%, at least0.2%, or at least 0.5% DHA.

In another embodiment, at least one plant part synthesizes at least oneω3 C20 LC-PUFA, wherein the total fatty acid of the plant part comprisesat least 2.5%, or at least 4.1% ω3 C20 LC-PUFA.

In yet another embodiment, at least one plant part synthesizes EPA,wherein the efficiency of conversion of ALA to EPA in the plant part isat least 2% or at least 14.6%.

In a further embodiment, at least one plant part synthesizes ω3polyunsaturated fatty acids that are the products of Δ6-desaturation ofALA and/or the products of Δ9 elongation of ALA, wherein the efficiencyof conversion of ALA to said products in the plant part is at least 22%or at least 24%.

In yet another embodiment, at least one plant part synthesizes DPA fromEPA, wherein the efficiency of conversion of EPA to DPA in the plantpart is at least 5% or at least 7%.

With regard to transgenic seeds of the invention, in one embodiment EPAis synthesized in the seed and the total fatty acid of the seedcomprises at least 1.5%, at least 2.1%, or at least 2.5% EPA.

In another embodiment, DPA is synthesized in the seed and the totalfatty acid of the seed comprises at least 0.1%, at least 0.13%, or atleast 0.5% DPA.

In a further embodiment, DHA is synthesized in the seed.

In another embodiment, DHA is synthesized in the seed and the totalfatty acid of the seed comprises at least 0.1%, at least 0.2%, or atleast 0.5% DHA.

In yet a further embodiment, at least one ω3 C20 LC-PUFA is synthesizedin the seed and the total fatty acid of the seed comprises at least2.5%, or at least 4.1% ω3 C20 LC-PUFA.

In a further embodiment. EPA is synthesized in the seed and theefficiency of conversion of ALA to EPA in the seed is at least 2% or atleast 14.6%.

In another embodiment, ω3 polyunsaturated fatty acids that are theproducts of Δ6-desaturation of ALA and/or the products of Δ9 elongationof ALA, are synthesized in the seed, and the efficiency of conversion ofALA to said products in the seed is at least 22% or at least 24%.

In a further embodiment, DPA is synthesized from EPA in the seed and theefficiency of conversion of EPA to DPA in the seed is at least 5% or atleast 7%.

Referring to extracts of the invention, in one embodiment, the totalfatty acid content of the extract comprises at least 1.5%, at least2.1%, or at least 2.5% EPA.

In another embodiment, the total fatty acid content of the extractcomprises at least 0.1%, at least 0.13%, or at least 0.5% DPA.

In a further embodiment, the extract comprises DHA.

In another embodiment, the total fatty acid content of the extractcomprises at east 0.1%, at least 0.2%, or at least 0.5% DHA.

In another embodiment, the total fatty acid content of the extractcomprises at least 2.5%, or at least 4.1% ω3 C20 LC-PUFA.

In yet a further embodiment, the extract comprises ARA, EPA, DPA, DHA,or any mixture of these in the triacylglycerols.

With regard to methods of the invention for producing a LC-PUFA, in onembodiment, the cell comprises at least one C20 LC-PUFA, and the totalfatty acid of the cell comprises at least 2%, at least 4.7%, or at least7.9% C20 LC-PUFA.

In another embodiment, the cell comprises at least one ω3 C20 LC-PUFA,and the total fatty acid of the cell comprises at least 2.5%, or atleast 4.1% ω3 C20 LC-PUFA.

In a further embodiment, the cell comprises ω3 polyunsaturated fattyacids that are the products of Δ6-desaturation of ALA and/or theproducts of Δ9 elongation of ALA, and the efficiency of conversion ofALA to said products in the cell is at least 22% or at least 24%.

In yet another embodiment, the cell comprises DPA, and the total fattyacid of the cell comprises at least 0.1%, at least 0.13%, or at least0.5% DPA.

In a further embodiment, the cell comprises DPA, and the efficiency ofconversion of EPA to DPA in the cell is at least 5% or at least 7%.

In another embodiment, the cell comprises EPA, and wherein the totalfatty acid of the cell comprises at least 1.5%, at least 2.1%, or atleast 2.5% EPA.

In a further embodiment, the cell comprises EPA, and the efficiency ofconversion of ALA to EPA in the cell is at least 2% or at least 14.6%.

Polypeptides

In one aspect, the present invention provides a substantially purifiedpolypeptide selected from the group consisting of:

i) a polypeptide comprising an amino acid sequence as provided in SEQ IDNO:1,

ii) a polypeptide comprising an amino acid sequence which is at least40% identical to SEQ ID NO:1, and

iii) a biologically active fragment of i) or ii),

wherein the polypeptide has Δ8 desaturase activity.

Preferably, the Δ8 desaturase does not also have Δ6 desaturase activity.

In another aspect, the present invention provides a substantiallypurified polypeptide selected from the group consisting of:

i) a polypeptide comprising an amino acid sequence as provided in SEQ IDNO:2,

ii) a polypeptide comprising an amino acid sequence which is at least60% identical to SEQ ID NO:2, and

iii) a biologically active fragment of i) or ii),

wherein the polypeptide has Δ5 elongase and/or Δ6 elongase activity.

Preferably, the polypeptide has Δ5 elongase and Δ6 elongase activity,and wherein the polypeptide is more efficient at synthesizing DPA fromEPA than it is at synthesizing ETA from SDA. More preferably, thepolypeptide can be purified from algae. Furthermore, when expressed inyeast cells, is more efficient at elongating C20 LC-PUFA than C22LC-PUFA.

In another aspect, the invention provides a substantially purifiedpolypeptide selected from the group consisting of:

i) a polypeptide comprising an amino acid sequence as provided in SEQ IDNO:3, SEQ ID NO:85 or SEQ ID NO:86,

ii) a polypeptide comprising an amino acid sequence which is at least40% identical to SEQ ID NO:3, SEQ ID NO:85 or SEQ ID NO:86, and

iii) a biologically active fragment of i) or ii),

wherein the polypeptide has Δ9 elongase and/or Δ6 elongase activity.

Preferably, the polypeptide has Δ9 elongase and Δ6 elongase activity.Preferably, the polypeptide is more efficient at synthesizing ETrA fromALA than it is at synthesizing ETA from SDA. Further, it is preferredthat the polypeptide can be purified from algae or fungi.

In yet another aspect, the present invention provides a substantiallypurified polypeptide selected from the group consisting of:

i) a polypeptide comprising an amino acid sequence as provided in SEQ IDNO:4,

ii) a polypeptide comprising an amino acid sequence which is at least70% identical to SEQ ID NO:4, and

iii) a biologically active fragment of i) or ii),

wherein the polypeptide has Δ4 desaturase activity.

In a further aspect, the present invention provides a substantiallypurified polypeptide selected from the group consisting of:

i) a polypeptide comprising an amino acid sequence as provided in SEQ IDNO:60,

ii) a polypeptide comprising an amino acid sequence which is at least55% identical to SEQ ID NO:60, and

iii) a biologically active fragment of i) or ii),

wherein the polypeptide has Δ5 desaturase activity.

In yet another aspect, the present invention provides a substantiallypurified polypeptide selected from the group consisting of:

i) a polypeptide comprising an amino acid sequence as provided in SEQ IDNO:64,

ii) a polypeptide comprising an amino acid sequence which is at least90% identical to SEQ ID NO:64, and

iii) a biologically active fragment of i) or ii),

wherein the polypeptide has Δ6 desaturase activity.

In yet another aspect, the present invention provides a substantiallypurified polypeptide selected from the group consisting of:

i) a polypeptide comprising an amino acid sequence as provided in SEQ IDNO:88,

ii) a polypeptide comprising an amino acid sequence which is at least76% identical to SEQ ID NO:88, and

iii) a biologically active fragment of i) or ii),

wherein the polypeptide has Δ6 elongase activity.

Preferably, in relation to any one of the above aspects, it is preferredthat the polypeptide can be isolated from a species selected from thegroup consisting of Pavlova and Melosira.

By “substantially purified polypeptide” we mean a polypeptide that hasbeen at least partially separated from the lipids, nucleic acids, otherpolypeptides, and other contaminating molecules with which it isassociated in its native state. Preferably, the substantially purifiedpolypeptide is at least 60% free, preferably at least 75% free, and mostpreferably at least 90% free from other components with which they arenaturally associated. Furthermore, the term “polypeptide” is usedinterchangeably herein with the term “protein”.

The % identity of a polypeptide is determined by GAP (Needleman andWunsch, 1970) analysis (GCG program) with a gap creation penalty=5, anda gap extension penalty=0.3. Unless stated otherwise, the query sequenceis at least 15 amino acids in length, and the GAP analysis aligns thetwo sequences over a region of at least 15 amino acids. More preferably,the query sequence is at least 50 amino acids in length, and the GAPanalysis aligns the two sequences over a region of at least 50 aminoacids. Even more preferably, the query sequence is at least 100 aminoacids in length and the GAP analysis aligns the two sequences over aregion of at least 100 amino acids.

With regard to the defined polypeptides/enzymes, it will be appreciatedthat % identity figures higher than those provided above will encompasspreferred embodiments. Thus, where applicable, in light of the minimum %identity figures, it is preferred that the polypeptide comprises anamino acid sequence which is at least 60%, more preferably at least 65%,more preferably at least 70%, more preferably at least 75%, morepreferably at least 76%, more preferably at least 80%, more preferablyat least 85%, more preferably at least 90%, more preferably at least91%, more preferably at least 92%, more preferably at least 93%, morepreferably at least 94%, more preferably at least 95%, more preferablyat least 96%, more preferably at least 97%, more preferably at least98%, more preferably at least 99%, more preferably at least 99.1%, morepreferably at least 99.2%, more preferably at least 99.3%, morepreferably at least 99.4%, more preferably at least 99.5%, morepreferably at least 99.6%, more preferably at least 99.7%, morepreferably at least 99.8%, and even more preferably at least 99.9%identical to the relevant nominated SEQ ID NO.

In a further embodiment, the present invention relates to polypeptideswhich are substantially identical to those specifically describedherein. As used herein, with reference to a polypeptide the term“substantially identical” means the deletion, insertion and/orsubstitution of one or a few (for example 2, 3, or 4) amino acids whilstmaintaining at least one activity of the native protein.

As used herein, the term “biologically active fragment” refers to aportion of the defined polypeptide/enzyme which still maintainsdesaturase or elongase activity (whichever is relevant). Suchbiologically active fragments can readily be determined by serialdeletions of the full length protein, and testing the activity of theresulting fragment.

Amino acid sequence mutants/variants of the polypeptides/enzymes definedherein can be prepared by introducing appropriate nucleotide changesinto a nucleic acid encoding the polypeptide, or by in vitro synthesisof the desired polypeptide. Such mutants include, for example,deletions, insertions or substitutions of residues within the amino acidsequence. A combination of deletion, insertion and substitution can bemade to arrive at the final construct, provided that the final proteinproduct possesses the desired characteristics.

In designing amino acid sequence mutants, the location of the mutationsite and the nature of the mutation will depend on characteristic(s) tobe modified. The sites for mutation can be modified individually or inseries, e.g., by (1) substituting first with conservative amino acidchoices and then with more radical selections depending upon the resultsachieved, (2) deleting the target residue, or (3) inserting otherresidues adjacent to the located site.

Amino acid sequence deletions generally range from about 1 to 30residues, more preferably about 1 to 10 residues and typically about 1to 5 contiguous residues.

Substitution mutants have at least one amino acid residue in thepolypeptide molecule removed and a different residue inserted in itsplace. The sites of greatest interest for substitutional mutagenesisinclude sites identified as the active or binding site(s). Other sitesof interest are those in which particular residues obtained from variousstrains or species are identical. These positions may be important forbiological activity. These sites, especially those falling within asequence of at least three other identically conserved sites, arepreferably substituted in a relatively conservative manner. Suchconservative substitutions are shown in Table 2.

Furthermore, if desired, unnatural amino acids or chemical amino acidanalogues can be introduced as a substitution or addition into thepolypeptides of the present invention. Such amino acids include, but arenot limited to, the D-isomers of the common amino acids,2,4-diaminobutyric acid, α-amino isobutyric acid, 4-aminobutyric acid,2-aminobutyric acid, 6-amino hexanoic acid, 2-amino isobutyric acid,3-amino propionic acid, ornithine, norleucine, norvaline,hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid,t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,β-alanine, fluoro-amino acids, designer amino acids such as β-methylamino acids, Cα-methyl amino acids, Nα-methyl amino acids, and aminoacid analogues in general.

Also included within the scope of the invention are polypeptides of thepresent invention which are differentially modified during or aftersynthesis, e.g., by biotinylation, benzylation, glycosylation,acetylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to an antibodymolecule or other cellular ligand, etc. These modifications may serve toincrease the stability and/or bioactivity of the polypeptide of theinvention.

TABLE 2 Exemplary substitutions. Original Exemplary ResidueSubstitutions Ala (A) val; leu; ile; gly Arg (R) lys Asn (N) gln; hisAsp (D) glu Cys (C) ser Gln (Q) asn; his Glu (E) asp Gly (G) pro, alaHis (H) asn; gln Ile (I) leu; val; ala Leu (L) ile; val; met; ala; pheLys (K) arg Met (M) leu; phe Phe (F) leu; val; ala Pro (P) gly Ser (S)thr Thr (T) ser Trp (W) tyr Tyr (Y) trp; phe Val (V) ile; leu; met; phe,ala

Polypeptides of the present invention can be produced in a variety ofways, including production and recovery of natural proteins, productionand recovery of recombinant proteins, and chemical synthesis of theproteins. In one embodiment, an isolated polypeptide of the presentinvention is produced by culturing a cell capable of expressing thepolypeptide under conditions effective to produce the polypeptide, andrecovering the polypeptide. A preferred cell to culture is a recombinantcell of the present invention. Effective culture conditions include, butare not limited to, effective media, bioreactor, temperature, pH andoxygen conditions that permit protein production. An effective mediumrefers to any medium in which a cell is cultured to produce apolypeptide of the present invention. Such medium typically comprises anaqueous medium having assimilable carbon, nitrogen and phosphatesources, and appropriate salts, minerals, metals and other nutrients,such as vitamins. Cells of the present invention can be cultured inconventional fermentation bioreactors, shake flasks, test tubes,microtiter dishes, and petri plates. Culturing can be carried out at atemperature, pH and oxygen content appropriate for a recombinant cell.Such culturing conditions are within the expertise of one of ordinaryskill in the art.

Polynucleotides

In one aspect, the present invention provides an isolated polynucleotidecomprising a sequence of nucleotides selected from the group consistingof:

i) a sequence of nucleotides as provided in SEQ ID NO:5 or SEQ ID NO:6;

ii) a sequence encoding a polypeptide of the invention;

iii) a sequence of nucleotides which is at least 50% identical to SEQ IDNO:5 or SEQ ID NO:6; and

iv) a sequence which hybridizes to any one of i) to iii) under highstringency conditions.

In another aspect, the present invention provides an isolatedpolynucleotide comprising a sequence of nucleotides selected from thegroup consisting of:

i) a sequence of nucleotides as provided in SEQ ID NO:7 or SEQ ID NO:8;

ii) a sequence encoding a polypeptide of the invention;

iii) a sequence of nucleotides which is at least 51% identical to SEQ IDNO:7 or SEQ ID NO:8; and

iv) a sequence which hybridizes to any one of i) to iii) under highstringency conditions.

In yet another aspect, the present invention provides an isolatedpolynucleotide comprising a sequence of nucleotides selected from thegroup consisting of:

i) a sequence of nucleotides as provided in SEQ ID NO:9 or SEQ ID NO:10;

ii) a sequence encoding a polypeptide of the invention;

iii) a sequence of nucleotides which is at least 51% identical to SEQ IDNO:9 or SEQ ID NO:10; and

iv) a sequence which hybridizes to any one of i) to iii) under highstringency conditions.

In a preferred embodiment, the sequence encoding a polypeptide of theinvention is nucleotides 31 to 915 or SEQ ID NO:9 or nucleotides 85 to915 of SEQ ID NO:9.

In a further aspect, the present invention provides an isolatedpolynucleotide comprising a sequence of nucleotides selected from thegroup consisting of:

i) a sequence of nucleotides as provided in SEQ ID NO:11, SEQ ID NO:12or SEQ ID NO:13;

ii) a sequence encoding a polypeptide of the invention;

iii) a sequence of nucleotides which is at least 70% identical to SEQ IDNO: 11, SEQ ID NO:12 or SEQ ID NO:13; and

iv) a sequence which hybridizes to any one of i) to iii) under highstringency conditions.

In another aspect, the present invention provides an isolatedpolynucleotide comprising a sequence of nucleotides selected from thegroup consisting of:

i) a sequence of nucleotides as provided in SEQ ID NO:58 or SEQ IDNO:59;

ii) a sequence encoding a polypeptide of the invention;

iii) a sequence of nucleotides which is at least 55% identical to SEQ IDNO:58 or SEQ ID NO:59; and

iv) a sequence which hybridizes to any one of i) to iii) under highstringency conditions.

In another aspect, the present invention provides an isolatedpolynucleotide comprising a sequence of nucleotides selected from thegroup consisting of:

i) a sequence of nucleotides as provided in SEQ ID NO:63;

ii) a sequence encoding a polypeptide of the invention;

iii) a sequence of nucleotides which is at least 90% identical to SEQ IDNO:63; and

iv) a sequence which hybridizes to any one of i) to iii) under highstringency conditions.

In another aspect, the present invention provides an isolatedpolynucleotide comprising a sequence of nucleotides selected from thegroup consisting of:

i) a sequence of nucleotides as provided in SEQ ID NO:89;

ii) a sequence encoding a polypeptide of the invention;

iii) a sequence of nucleotides which is at least 76% identical to SEQ IDNO:89; and

iv) a sequence which hybridizes to any one of i) to iii) under highstringency conditions.

The present inventors are also the first to isolate polynucleotideencoding a keto-acyl synthase-like fatty acid elongase from a non-higherplant.

Accordingly, in a further aspect the present invention provides anisolated polynucleotide comprising a sequence of nucleotides selectedfrom the group consisting of:

i) a sequence of nucleotides as provided in SEQ ID NO:55;

ii) a sequence of nucleotides which is at least 40% identical to SEQ IDNO:55; and

iii) a sequence which hybridizes to i) or ii) under high stringencyconditions.

By an “isolated polynucleotide”, including DNA, RNA, or a combination ofthese, single or double stranded, in the sense or antisense orientationor a combination of both, dsRNA or otherwise, we mean a polynucleotidewhich is at least partially separated from the polynucleotide sequenceswith which it is associated or linked in its native state. Preferably,the isolated polynucleotide is at least 60% free, preferably at least75% free, and most preferably at least 90% free from other componentswith which they are naturally associated. Furthermore, the term“polynucleotide” is used interchangeably herein with the term “nucleicacid molecule”.

The % identity of a polynucleotide is determined by GAP (Needleman andWunsch, 1970) analysis (GCG program) with a gap creation penalty=5, anda gap extension penalty=0.3. Unless stated otherwise, the query sequenceis at least 45 nucleotides in length, and the GAP analysis aligns thetwo sequences over a region of at least 45 nucleotides. Preferably, thequery sequence is at least 150 nucleotides in length, and the GAPanalysis aligns the two sequences over a region of at least 150nucleotides. More preferably, the query sequence is at least 300nucleotides in length and the GAP analysis aligns the two sequences overa region of at least 300 nucleotides.

With regard to the defined polynucleotides, it will be appreciated that% identity figures higher than those provided above will encompasspreferred embodiments. Thus, where applicable, in light of the minimum %identity figures, it is preferred that the polynucleotide comprises anucleotide sequence which is at least 60%, more preferably at least 65%,more preferably at least 70%, more preferably at least 75%, morepreferably at least 76%, more preferably at least 80%, more preferablyat least 85%, more preferably at least 90%, more preferably at least91%, more preferably at least 92%, more preferably at least 93%, morepreferably at least 94%, more preferably at least 95%, more preferablyat least 96%, more preferably at least 97%, more preferably at least98%, more preferably at least 99%, more preferably at least 99.1%, morepreferably at least 99.2%, more preferably at least 99.3%, morepreferably at least 99.4%, more preferably at least 99.5%, morepreferably at least 99.6%, more preferably at least 99.7%, morepreferably at least 99.8%, and even more preferably at least 99.9%identical to the relevant nominated SEQ ID NO.

In a further embodiment, the present invention relates topolynucleotides which are substantially identical to those specificallydescribed herein. As used herein, with reference to a polynucleotide theterm “substantially identical” means the substitution of one or a few(for example 2, 3, or 4) nucleotides whilst maintaining at least oneactivity of the native protein encoded by the polynucleotide. Inaddition, this term includes the addition or deletion of nucleotideswhich results in the increase or decrease in size of the encoded nativeprotein by one or a few (for example 2, 3, or 4) amino acids whilstmaintaining at least one activity of the native protein encoded by thepolynucleotide.

Oligonucleotides of the present invention can be RNA. DNA, orderivatives of either. The minimum size of such oligonucleotides is thesize required for the formation of a stable hybrid between anoligonucleotide and a complementary sequence on a nucleic acid moleculeof the present invention. Preferably, the oligonucleotides are at least15 nucleotides, more preferably at least 18 nucleotides, more preferablyat least 19 nucleotides, more preferably at least 20 nucleotides, evenmore preferably at least 25 nucleotides in length. The present inventionincludes oligonucleotides that can be used as, for example, probes toidentify nucleic acid molecules, or primers to produce nucleic acidmolecules. Oligonucleotide of the present invention used as a probe aretypically conjugated with a label such as a radioisotope, an enzyme,biotin, a fluorescent molecule or a chemiluminescent molecule.

Polynucleotides and oligonucleotides of the present invention includethose which hybridize under stringent conditions to a sequence providedas SEQ ID NO's: 5 to 13. As used herein, stringent conditions are thosethat (1) employ low ionic strength and high temperature for washing, forexample, 0.015 M NaCl/0.0015 M sodium citrate/0.1% NaDodSO₄ at 50° C.;(2) employ during hybridisation a denaturing agent such as formamide,for example, 50% (vol/vol) form amide with 0.1% bovine serum albumin,0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer atpH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42° C.; or (3) employ50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 g/ml), 0.1% SDS and 10% dextran sulfateat 42° C. in 0.2×SSC and 0.1% SDS.

Polynucleotides of the present invention may possess, when compared tonaturally occurring molecules, one or more mutations which aredeletions, insertions, or substitutions of nucleotide residues. Mutantscan be either naturally occurring (that is to say, isolated from anatural source) or synthetic (for example, by performing site-directedmutagenesis on the nucleic acid).

Also provided are antisense and/or catalytic nucleic acids (such asribozymes) which hybridize to a polynucleotide of the invention, andhence inhibit the production of an encoded protein. Furthermore,provided are dsRNA molecules, particularly small dsRNA molecules with adouble stranded region of about 21 nucleotides, which can be used in RNAinterference to inhibit the production of a polypeptide of the inventionin a cell. Such inhibitory molecules can be used to alter the types offatty acids produced by a cell, such an animal cell, moss, or algaelcell. The production of such antisense, catalytic nucleic acids anddsRNA molecules is well with the capacity of the skilled person (see forexample, G. Hartmann and S. Endres, Manual of Antisense Methodology,Kluwer (1999); Haseloff and Gerlach, 1988; Perriman et al., 1992; Shippyet al., 1999; Waterhouse et al. (1998); Smith et al. (2000); WO99/32619, WO 99/53050, WO 99/49029, and WO 01/34815).

Gene Constructs and Vectors

One embodiment of the present invention includes a recombinant vector,which includes at least one isolated polynucleotide molecule encoding apolypeptide/enzyme defined herein, inserted into any vector capable ofdelivering the nucleic acid molecule into a host cell. Such a vectorcontains heterologous nucleic acid sequences, that is nucleic acidsequences that are not naturally found adjacent to nucleic acidmolecules of the present invention and that preferably are derived froma species other than the species from which the nucleic acid molecule(s)are derived. The vector can be either RNA or DNA, either prokaryotic oreukaryotic, and typically is a virus or a plasmid.

One type of recombinant vector comprises a nucleic acid molecule of thepresent invention operatively linked to an expression vector. Asindicated above, the phrase operatively linked refers to insertion of anucleic acid molecule into an expression vector in a manner such thatthe molecule is able to be expressed when transformed into a host cell.As used herein, an expression vector is a DNA or RNA vector that iscapable of transforming a host cell and effecting expression of aspecified nucleic acid molecule. Preferably, the expression vector isalso capable of replicating within the host cell. Expression vectors canbe either prokaryotic or eukaryotic, and are typically viruses orplasmids. Expression vectors of the present invention include anyvectors that function (i.e., direct gene expression) in recombinantcells of the present invention, including in bacterial, fungal,endoparasite, arthropod, other animal, and plant cells. Preferredexpression vectors of the present invention can direct gene expressionin yeast, animal or plant cells.

In particular, expression vectors of the present invention containregulatory sequences such as transcription control sequences,translation control sequences, origins of replication, and otherregulatory sequences that are compatible with the recombinant cell andthat control the expression of nucleic acid molecules of the presentinvention. In particular, recombinant molecules of the present inventioninclude transcription control sequences. Transcription control sequencesare sequences which control the initiation, elongation, and terminationof transcription. Particularly important transcription control sequencesare those which control transcription initiation, such as promoter,enhancer, operator and repressor sequences. Suitable transcriptioncontrol sequences include any transcription control sequence that canfunction in at least one of the recombinant cells of the presentinvention. A variety of such transcription control sequences are knownto those skilled in the art.

Another embodiment of the present invention includes a recombinant cellcomprising a host cell transformed with one or more recombinantmolecules of the present invention. Transformation of a nucleic acidmolecule into a cell can be accomplished by any method by which anucleic acid molecule can be inserted into the cell. Transformationtechniques include, but are not limited to, transfection,electroporation, microinjection, lipofection, adsorption, and protoplastfusion. A recombinant cell may remain unicellular or may grow into atissue, organ or a multicellular organism. Transformed nucleic acidmolecules can remain extrachromosomal or can integrate into one or moresites within a chromosome of the transformed (i.e., recombinant) cell insuch a manner that their ability to be expressed is retained.

Transgenic Plants and Parts Thereof

The term “plant” as used herein as a noun refers to whole plants, but asused as an adjective refers to any substance which is present in,obtained from, derived from, or related to a plant, such as for example,plant organs (e.g. leaves, stems, roots, flowers), single cells (e.g.pollen), seeds, plant cells and the like. Plants provided by orcontemplated for use in the practice of the present invention includeboth monocotyledons and dicotyledons. In preferred embodiments, theplants of the present invention are crop plants (for example, cerealsand pulses, maize, wheat, potatoes, tapioca, rice, sorghum, millet,cassava, barley, or pea), or other legumes. The plants may be grown forproduction of edible roots, tubers, leaves, stems, flowers or fruit. Theplants may be vegetables or ornamental plants. The plants of theinvention may be: corn (Zea mays), canola (Brassica napus, Brassica rapassp.), flax (Linum usitatissimum), alfalfa (Medicago sativa), rice(Oryza sativa), rye (Secale cerale), sorghum (Sorghum bicolour, Sorghumvulgar), sunflower (Helianthus annus), wheat (Tritium aestivum), soybean(Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum),peanuts (Arachis hypogaea), cotton (Gossypium hirsutum), sweet potato(Lopmoea batatus), cassava (Manihot esculenta), coffee (Cofea spp.),coconut (Cocos nucifera), pineapple (Anana comosus), citris tree (Citrusspp.), cocoa (Theobroma cacao), tea (Camellia senensis), banana (Musaspp.), avocado (Persea americana), fig (Ficus casica), guava (Psidiumguajava), mango (Mangifer indica), olive (Olea europaea), papaya (Caricapapaya), cashew (Anacardium occidentale), macadamia (Macadamiaintergrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris),oats, or barley.

In one embodiment, the plant is an oilseed plant, preferably an oilseedcrop plant. As used herein, an “oilseed plant” is a plant species usedfor the commercial production of oils from the seeds of the plant. Theoilseed plant may be oil-seed rape (such as canola), maize, sunflower,soybean, sorghum, flax (linseed) or sugar beet. Furthermore, the oilseedplant may be other Brassicas, cotton, peanut, poppy, mustard, castorbean, sesame, safflower, or nut producing plants. The plant may producehigh levels of oil in its fruit, such as olive, oil palm or coconut.Horticultural plants to which the present invention may be applied arelettuce, endive, or vegetable brassicas including cabbage, broccoli, orcauliflower. The present invention may be applied in tobacco, cucurbits,carrot, strawberry, tomato, or pepper.

When the production of ω3 LC-PUFA is desired it is preferable that theplant species which is to be transformed has an endogenous ratio of ALAto LA which is at least 1:1, more preferably at least 2:1. Examplesinclude most, if not all, oilseeds such as linseed. This maximizes theamount of ALA substrate available for the production of SDA, ETA, ETrA,EPA, DPA and DHA.

The plants produced using the methods of the invention may already betransgenic, and/or transformed with additional genes to those describedin detail herein. In one embodiment, the transgenic plants of theinvention also produce a recombinant ω3 desaturase. The presence of arecombinant ω3 desaturase increases the ratio of ALA to LA in the plantswhich, as outlined in the previous paragraph, maximizes the productionof LC-PUFA such as SDA, ETA, ETrA, EPA, DPA and DHA.

Grain plants that provide seeds of interest include oil-seed plants andleguminous plants. Seeds of interest include grain seeds, such as corn,wheat, barley, rice, sorghum, rye, etc. Leguminous plants include beansand peas. Beans include guar, locust bean, fenugreek, soybean, gardenbeans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc.

The term “extract or portion thereof” refers to any part of the plant.“Portion” generally refers to a specific tissue or organ such as a seedor root, whereas an “extract” typically involves the disruption of cellwalls and possibly the partial purification of the resulting material.Naturally, the “extract or portion thereof” will comprise at least oneLC-PUFA. Extracts can be prepared using standard techniques of the art.

Transgenic plants, as defined in the context of the present inventioninclude plants and their progeny which have been genetically modifiedusing recombinant techniques. This would generally be to cause orenhance production of at least one protein/enzyme defined herein in thedesired plant or plant organ. Transgenic plant parts include all partsand cells of said plants such as, for example, cultured tissues, callus,protoplasts. Transformed plants contain genetic material that they didnot contain prior to the transformation. The genetic material ispreferably stably integrated into the genome of the plant. Theintroduced genetic material may comprise sequences that naturally occurin the same species but in a rearranged order or in a differentarrangement of elements, for example an antisense sequence. Such plantsare included herein in “transgenic plants”. A “non-transgenic plant” isone which has not been genetically modified with the introduction ofgenetic material by recombinant DNA techniques. In a preferredembodiment, the transgenic plants are homozygous for each and every genethat has been introduced (transgene) so that their progeny do notsegregate for the desired phenotype.

Several techniques exist for introducing foreign genetic material into aplant cell. Such techniques include acceleration of genetic materialcoated onto microparticle* directly into cells (see, for example, U.S.Pat. No. 4,945,050 and U.S. Pat. No. 5,141,131). Plants may betransformed using Agrobacterium technology (see, for example, U.S. Pat.No. 5,177,010, U.S. Pat. No. 5,104,310, U.S. Pat. No. 5,004,863, U.S.Pat. No. 5,159,135). Electroporation technology has also been used totransform plants (see, for example, WO 87/06614, U.S. Pat. Nos.5,472,869, 5,384,253, WO 92/09696 and WO 93/21335). In addition tonumerous technologies for transforming plants, the type of tissue whichis contacted with the foreign genes may vary as well. Such tissue wouldinclude but would not be limited to embryogenic tissue, callus tissuetype I and II, hypocotyl, meristem, and the like. Almost all planttissues may be transformed during development and/or differentiationusing appropriate techniques described herein.

A number of vectors suitable for stable transfection of plant cells orfor the establishment of transgenic plants have been described in, e.g.,Pouwels et al. Cloning Vectors: A Laboratory Manual, 1985, supp. 1987;Weissbach and Weissbach, Methods for Plant Molecular Biology, AcademicPress, 1989; and Gelvin et al., Plant Molecular Biology Manual, KluwerAcademic Publishers, 1990. Typically, plant expression vectors include,for example, one or more cloned plant genes under the transcriptionalcontrol of 5′ and 3′ regulatory sequences and a dominant selectablemarker. Such plant expression vectors also can contain a promoterregulatory region (e.g., a regulatory region controlling inducible orconstitutive, environmentally- or developmentally-regulated, or cell- ortissue-specific expression), a transcription initiation start site, aribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal.

Examples of plant promoters include, but are not limited toribulose-1,6-bisphosphate carboxylase small subunit, beta-conglycininpromoter, phaseolin promoter, high molecular weight glutenin (HMW-GS)promoters, starch biosynthetic gene promoters, ADH promoter, heat-shockpromoters and tissue specific promoters. Promoters may also containcertain enhancer sequence elements that may improve the transcriptionefficiency. Typical enhancers include but are not limited to Adh-intronI and Adh-intron 6.

Constitutive promoters direct continuous gene expression in all cellstypes and at all times (e.g., actin, ubiquitin. CaMV 35S). Tissuespecific promoters are responsible for gene expression in specific cellor tissue types, such as the leaves or seeds (e.g., zein, oleosin,napin. ACP, globulin and the like) and these promoters may also be used.Promoters may also be active during a certain stage of the plants'development as well as active in plant tissues and organs. Examples ofsuch promoters include but are not limited to pollen-specific, embryospecific, corn silk specific, cotton fibre specific, root specific, seedendosperm specific promoters and the like.

In a particularly preferred embodiment, the promoter directs expressionin tissues and organs in which lipid and oil biosynthesis take place,particularly in seed cells such as endosperm cells and cells of thedeveloping embryo. Promoters which are suitable are the oilseed rapenapin gene promoter (U.S. Pat. No. 5,608,152), the Vicia faba USPpromoter (Baumlein et al., 1991), the Arabidopsis oleosin promoter (WO98/45461), the Phaseolus vulgaris phaseolin promoter (U.S. Pat. No.5,504,200), the Brassica Bce4 promoter (WO 91/13980) or the legumin B4promoter (Baumlein et al., 1992), and promoters which lead to theseed-specific expression in monocots such as maize, barley, wheat, rye,rice and the like. Notable promoters which are suitable are the barleyIpt2 or Ipt1 gene promoter (WO 95/15389 and WO 95/23230) or thepromoters described in WO 99/16890 (promoters from the barley hordeingene, the rice glutelin gene, the rice oryzin gene, the rice prolamingene, the wheat gliadin gene, the wheat glutelin gene, the maize zeingene, the oat glutelin gene, the sorghum kasirin gene, the rye secalingene). Other promoters include those described by Broun et al. (1998)and US 20030159173.

Under certain circumstances it may be desirable to use an induciblepromoter. An inducible promoter is responsible for expression of genesin response to a specific signal, such as: physical stimulus (heat shockgenes); light (RUBP carboxylase); hormone (Em); metabolites; and stress.Other desirable transcription and translation elements that function inplants may be used.

In addition to plant promoters, promoters from a variety of sources canbe used efficiently in plant cells to express foreign genes. Forexample, promoters of bacterial origin, such as the octopine synthasepromoter, the nopaline synthase promoter, the mannopine synthasepromoter; promoters of viral origin, such as the cauliflower mosaicvirus (35S and 19S) and the like may be used.

It will be apparent that transgenic plants adapted for the production ofLC-PUFA as described herein, in particular DHA, can either be eatendirectly or used as a source for the extraction of essential fattyacids, of which DHA would be a constituent.

As used herein, “germination” refers to the emergence of the root tipfrom the seed coat after imbibition. “Germination rate” refers to thepercentage of seeds in a population which have germinated over a periodof time, for example 7 or 10 days, after imbibition. A population ofseeds can be assessed daily over several days to determine thegermination percentage over time.

With regard to seeds of the present invention, as used herein the term“germination rate which is substantially the same” means that thegermination rate of the transgenic seeds is at least 60%, morepreferably at least 80%, and even more preferably at least 90%, that ofisogenic non-transgenic seeds. Gemination rates can be calculated usingtechniques known in the art.

With further regard to seeds of the present invention, as used hereinthe term “timing of germination of the seed is substantially the same”means that the timing of germination of the transgenic seeds is at least60%, more preferably at least 80%, and even more preferably at least90%, that of isogenic non-transgenic seeds. Timing of gemination can becalculated using techniques known in the art.

The present inventors have found that at least in some circumstances theproduction of LC-PUFA in recombinant plant cells is enhanced when thecells are homozygous for the transgene. As a result, it is preferredthat the recombinant plant cell, preferably the transgenic plant, ishomozygous for at least one desaturase and/or elongase gene. In oneembodiment, the cells/plant are homozygous for the zebrafish Δ6/Δ5desaturase and/or the C. elegans elongase.

Transgenic Non-Human Animals

Techniques for producing transgenic animals are well known in the art. Auseful general textbook on this subject is Houdebine. Transgenicanimals—Generation and Use (Harwood Academic, 1997).

Heterologous DNA can be introduced, for example, into fertilizedmammalian ova. For instance, totipotent or pluripotent stem cells can betransformed by microinjection, calcium phosphate mediated precipitation,liposome fusion, retroviral infection or other means, the transformedcells are then introduced into the embryo, and the embryo then developsinto a transgenic animal. In a highly preferred method, developingembryos are infected with a retrovirus containing the desired DNA, andtransgenic animals produced from the infected embryo. In a mostpreferred method, however, the appropriate DNAs are coinjected into thepronucleus or cytoplasm of embryos, preferably at the single cell stage,and the embryos allowed to develop into mature transgenic animals.

Another method used to produce a transgenic animal involvesmicroinjecting a nucleic acid into pro-nuclear stage eggs by standardmethods. Injected eggs are then cultured before transfer into theoviducts of pseudopregnant recipients.

Transgenic animals may also be produced by nuclear transfer technology.Using this method, fibroblasts from donor animals are stably transfectedwith a plasmid incorporating the coding sequences for a binding domainor binding partner of interest under the control of regulatory. Stabletransfectants are then fused to enucleated oocytes, cultured andtransferred into female recipients.

Feedstuffs

The present invention includes compositions which can be used asfeedstuffs. For purposes of the present invention, “feedstuffs” includeany food or preparation for human or animal consumption (including forenteral and/or parenteral consumption) which when taken into the body(a) serve to nourish or build up tissues or supply energy; and/or (b)maintain, restore or support adequate nutritional status or metabolicfunction. Feedstuffs of the invention include nutritional compositionsfor babies and/or young children.

Feedstuffs of the invention comprise, for example, a cell of theinvention, a plant of the invention, the plant part of the invention,the seed of the invention, an extract of the invention, the product ofthe method of the invention, the product of the fermentation process ofthe invention, or a composition along with a suitable carrier(s). Theterm “carrier” is used in its broadest sense to encompass any componentwhich may or may not have nutritional value. As the skilled addresseewill appreciate, the carrier must be suitable for use (or used in asufficiently low concentration) in a feedstuff such that it does nothave deleterious effect on an organism which consumes the feedstuff.

The feedstuff of the present invention comprises an oil, fatty acidester, or fatty acid produced directly or indirectly by use of themethods, cells or plants disclosed herein. The composition may either bein a solid or liquid form. Additionally, the composition may includeedible macronutrients, vitamins, and/or minerals in amounts desired fora particular use. The amounts of these ingredients will vary dependingon whether the composition is intended for use with normal individualsor for use with individuals having specialized needs, such asindividuals suffering from metabolic disorders and the like.

Examples of suitable carriers with nutritional value include, but arenot limited to, macronutrients such as edible fats, carbohydrates andproteins. Examples of such edible fats include, but are not limited to,coconut oil, borage oil, fungal oil, black current oil, soy oil, andmono- and diglycerides. Examples of such carbohydrates include (but arenot limited to): glucose, edible lactose, and hydrolyzed search.Additionally, examples of proteins which may be utilized in thenutritional composition of the invention include (but are not limitedto) soy proteins, electrodialysed whey, electrodialysed skim milk, milkwhey, or the hydrolysates of these proteins.

With respect to vitamins and minerals, the following may be added to thefeedstuff compositions of the present invention: calcium, phosphorus,potassium, sodium, chloride, magnesium, manganese, iron, copper, zinc,selenium, iodine, and Vitamins A, E, D, C, and the B complex. Other suchvitamins and minerals may also be added.

The components utilized in the feedstuff compositions of the presentinvention can be of semi-purified or purified origin. By semi-purifiedor purified is meant a material which has been prepared by purificationof a natural material or by de novo synthesis.

A feedstuff composition of the present invention may also be added tofood even when supplementation of the diet is not required. For example,the composition may be added to food of any type, including (but notlimited to): margarine, modified butter, cheeses, milk, yogurt,chocolate, candy, snacks, salad oils, cooking oils, cooking fats, meats,fish and beverages.

The genus Saccharomyces spp is used in both brewing of beer and winemaking and also as an agent in baking, particularly bread. Yeast is amajor constituent of vegetable extracts. Yeast is also used as anadditive in animal feed. It will be apparent that genetically engineeredyeast strains can be provided which are adapted to synthesise LC-PUFA asdescribed herein. These yeast strains can then be used in food stuffsand in wine and beer making to provide products which have enhancedfatty acid content and in particular DHA content.

Additionally, LC-PUFA produced in accordance with the present inventionor host cells transformed to contain and express the subject genes mayalso be used as animal food supplements to alter an animal's tissue ormilk fatty acid composition to one more desirable for human or animalconsumption. Examples of such animals include sheep, cattle, horses andthe like.

Furthermore, feedstuffs of the invention can be used in aquaculture toincrease the levels of LC-PUFA in fish for human or animal consumption.

In mammals, the so-called “Sprecher” pathway converts DPA to DHA bythree reactions, independent of a Δ7 elongase, Δ4 desaturase, and abeta-oxidation step (Sprecher et al., 1995) (FIG. 1). Thus, infeedstuffs for mammal consumption, for example formulations for theconsumption by human infants, it may only be necessary to provide DPAproduced using the methods of the invention as the mammalian subjectshould be able to fulfill its nutritional needs for DHA by using the“Sprecher” pathway to convert DPA to DHA. As a result, in an embodimentof the present invention, a feedstuff described herein for mammalianconsumption at least comprises DPA, wherein at least one enzymaticreaction in the production of DPA was performed by a recombinant enzymein a cell.

Compositions

The present invention also encompasses compositions, particularlypharmaceutical compositions, comprising one or more of the fatty acidsand/or resulting oils produced using the methods of the invention.

A pharmaceutical composition may comprise one or more of the LC-PUFAand/or oils, in combination with a standard, well-known, non-toxicpharmaceutically-acceptable carrier, adjuvant or vehicle such asphosphate-buffered saline, water, ethanol, polyols, vegetable oils, awetting agent or an emulsion such as a water/oil emulsion. Thecomposition may be in either a liquid or solid form. For example, thecomposition may be in the form of a tablet, capsule, ingestible liquidor powder, injectable, or topical ointment or cream. Proper fluidity canbe maintained, for example, by the maintenance of the required particlesize in the case of dispersions and by the use of surfactants. It mayalso be desirable to include isotonic agents, for example, sugars,sodium chloride, and the like. Besides such inert diluents, thecomposition can also include adjuvants, such as wetting agents,emulsifying and suspending agents, sweetening agents, flavoring agentsand perfuming agents.

Suspensions, in addition to the active compounds, may comprisesuspending agents such as ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanthor mixtures of these substances.

Solid dosage forms such as tablets and capsules can be prepared usingtechniques well known in the art. For example, LC-PUFA produced inaccordance with the present invention can be tableted with conventionaltablet bases such as lactose, sucrose, and cornstarch in combinationwith binders such as acacia, cornstarch or gelatin, disintegratingagents such as potato starch or alginic acid, and a lubricant such asstearic acid or magnesium stearate. Capsules can be prepared byincorporating these excipients into a gelatin capsule along withantioxidants and the relevant LC-PUFA(s).

For intravenous administration, the PUFA produced in accordance with thepresent invention or derivatives thereof may be incorporated intocommercial formulations.

A typical dosage of a particular fatty acid is from 0.1 mg to 20 g,taken from one to five times per day (up to 100 g daily) and ispreferably in the range of from about 10 mg to about 1, 2, 5, or 10 gdaily (taken in one or multiple doses). As known in the art, a minimumof about 300 mg/day of LC-PUFA is desirable. However, it will beappreciated that any amount of LC-PUFA will be beneficial to thesubject.

Possible routes of administration of the pharmaceutical compositions ofthe present invention include, for example, enteral (e.g., oral andrectal) and parenteral. For example, a liquid preparation may beadministered orally or rectally. Additionally, a homogenous mixture canbe completely dispersed in water, admixed under sterile conditions withphysiologically acceptable diluents, preservatives, buffers orpropellants to form a spray or inhalant.

The dosage of the composition to be administered to the patient may bedetermined by one of ordinary skill in the art and depends upon variousfactors such as weight of the patient, age of the patient, overallhealth of the patient, past history of the patient, immune status of thepatient, etc.

Additionally, the compositions of the present invention may be utilizedfor cosmetic purposes. It may be added to pre-existing cosmeticcompositions such that a mixture is formed or a LC-PUFA producedaccording to the subject invention may be used as the sole “active”ingredient in a cosmetic composition.

Medical, Veterinary, Agricultural and Aquacultural Uses

The present invention also includes the treatment of various disordersby use of the pharmaceutical and/or feedstuff compositions describedherein. In particular, the compositions of the present invention may beused to treat restenosis after angioplasty. Furthermore, symptoms ofinflammation, rheumatoid arthritis, asthma and psoriasis may also betreated with the compositions (including feedstuffs) of the invention.Evidence also indicates that LC-PUFA may be involved in calciummetabolism; thus, the compositions of the present invention may beutilized in the treatment or prevention of osteoporosis and of kidney orurinary tract stones.

Additionally, the compositions of the present invention may also be usedin the treatment of cancer. Malignant cells have been shown to havealtered fatty acid compositions. Addition of fatty acids has been shownto slow their growth, cause cell death and increase their susceptibilityto chemotherapeutic agents. Moreover, the compositions of the presentinvention may also be useful for treating cachexia associated withcancer.

The compositions of the present invention may also be used to treatdiabetes as altered fatty acid metabolism and composition have beendemonstrated in diabetic animals.

Furthermore, the compositions of the present invention, comprisingLC-PUFA produced either directly or indirectly through the use of thecells of the invention, may also be used in the treatment of eczema andin the reduction of blood pressure. Additionally, the compositions ofthe present invention may be used to inhibit platelet aggregation, toinduce vasodilation, to reduce cholesterol levels, to inhibitproliferation of vessel wall smooth muscle and fibrous tissue, to reduceor to prevent gastrointestinal bleeding and other side effects ofnon-steroidal anti-inflammatory drugs (U.S. Pat. No. 4,666,701), toprevent or to treat endometriosis and premenstrual syndrome (U.S. Pat.No. 4,758,592), and to treat myalgic encephalomyelitis and chronicfatigue after viral infections (U.S. Pat. No. 5,116,871).

Further uses of the compositions of the present invention include, butare not limited to, use in the treatment or prevention of cardiacarrhythmia's, angioplasty, AIDS, multiple sclerosis, Crohn's disease,schizophrenia, foetal alcohol syndrome, attention deficienthyperactivity disorder, cystic fibrosis, phenylketonuria, unipolardepression, aggressive hostility, adrenoleukodystophy, coronary heartdisease, hypertension, obesity, Alzheimer's disease, chronic obstructivepulmonary disease, ulcerative colitis or an ocular disease, as well asfor maintenance of general health.

Furthermore, the above-described pharmaceutical and nutritionalcompositions may be utilized in connection with animals (i.e., domesticor non-domestic, including mammals, birds, reptiles, lizards, etc.), aswell as humans, as animals experience many of the same needs andconditions as humans. For example, the oil or fatty acids of the presentinvention may be utilized in animal feed supplements, animal feedsubstitutes, animal vitamins or in animal topical ointments.

Compositions such as feedstuffs of the invention can also be used inaquaculture to increase the levels of LC-PUFA in fish for human oranimal consumption.

Any amount of LC-PUFA will be beneficial to the subject. However, it ispreferred that an “amount effective to treat” the condition of interestis administered to the subject. Such dosages to effectively treat acondition which would benefit from administration of a LC-PUFA are knownthose skilled in the art. As an example, a dose of at least 300 mg/dayof LC-PUFA for at least a few weeks, more preferably longer would besuitable in many circumstances.

Antibodies

The invention also provides monoclonal and/or polyclonal antibodieswhich bind specifically to at least one polypeptide of the invention ora fragment thereof. Thus, the present invention further provides aprocess for the production of monoclonal or polyclonal antibodies topolypeptides of the invention.

The term “binds specifically” refers to the ability of the antibody tobind to at least one protein of the present invention but not otherproteins present in a recombinant cell, particularly a recombinant plantcell, of the invention.

As used herein, the term “epitope” refers to a region of a protein ofthe invention which is bound by the antibody. An epitope can beadministered to an animal to generate antibodies against the epitope,however, antibodies of the present invention preferably specificallybind the epitope region in the context of the entire protein.

If polyclonal antibodies are desired, a selected mammal (e.g., mouse,rabbit, goat, horse, etc.) is immunised with an immunogenic polypeptide.Serum from the immunised animal is collected and treated according toknown procedures. If serum containing polyclonal antibodies containsantibodies to other antigens, the polyclonal antibodies can be purifiedby immunoaffinity chromatography. Techniques for producing andprocessing polyclonal antisera are known in the art. In order that suchantibodies may be made, the invention also provides polypeptides of theinvention or fragments thereof haptenised to another polypeptide for useas immunogens in animals or humans.

Monoclonal antibodies directed against polypeptides of the invention canalso be readily produced by one skilled in the art. The generalmethodology for making monoclonal antibodies by hybridomas is wellknown. Immortal antibody-producing cell lines can be created by cellfusion, and also by other techniques such as direct transformation of Blymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus.Panels of monoclonal antibodies produced can be screened for variousproperties; i.e., for isotype and epitope affinity.

An alternative technique involves screening phage display librarieswhere, for example the phage express scFv fragments on the surface oftheir coat with a large variety of complementarity determining regions(CDRs). This technique is well known in the art.

For the purposes of this invention, the term “antibody”, unlessspecified to the contrary, includes fragments of whole antibodies whichretain their binding activity for a target antigen. Such fragmentsinclude Fv, F(ab′) and F(ab′)₂ fragments, as well as single chainantibodies (scFv). Furthermore, the antibodies and fragments thereof maybe humanised antibodies, for example as described in EP-A-239400.

Antibodies of the invention may be bound to a solid support and/orpackaged into kits in a suitable container along with suitable reagents,controls, instructions and the like.

Preferably, antibodies of the present invention are detectably labeled.Exemplary detectable labels that allow for direct measurement ofantibody binding include radiolabels, fluorophores, dyes, magneticbeads, chemiluminescers, colloidal particles, and the like. Examples oflabels which permit indirect measurement of binding include enzymeswhere the substrate may provide for a coloured or fluorescent product.Additional exemplary detectable labels include covalently bound enzymescapable of providing a detectable product signal after addition ofsuitable substrate. Examples of suitable enzymes for use in conjugatesinclude horseradish peroxidase, alkaline phosphatase, malatedehydrogenase and the like. Where not commercially available, suchantibody-enzyme conjugates are readily produced by techniques known tothose skilled in the art. Further exemplary detectable labels includebiotin, which binds with high affinity to avidin or streptavidin;fluorochromes (e.g., phycobiliproteins, phycoerythrin andallophycocyanins; fluorescein and Texas red), which can be used with afluorescence activated cell sorter; haptens; and the like. Preferably,the detectable label allows for direct measurement in a plateluminometer, e.g., biotin. Such labeled antibodies can be used intechniques known in the art to detect proteins of the invention.

EXAMPLES Example 1 Materials and Methods

Culturing Pavlova salina

Pavlova salina isolates including strain CS-49 from the CSIRO Collectionof Living Microalgae was cultivated under standard culture conditions(http://www.marine.csiro.au/microalgae). A stock culture from theCollection was sub-cultured and scaled-up in a dilution of 1 in 10 overconsecutive transfers in 1 L Erlenmeyer flasks and then into 10 Lpolycarbonate carboys. The culture medium was f/2, a modification ofGuillard and Ryther's (1962) f medium containing half-strengthnutrients, with a growth temperature of 20±1° C. Other culturingconditions included a light intensity of 100 μmol. photons PAR·m⁻²·s⁻¹,12:12 hour light:dark photoperiod, and bubbling with 1% CO₂ in air at arate of 200 mL·L⁻¹·min⁻.

Yeast Culturing and Feeding with Cursor Fact Acids

Plasmids were introduced into yeast by heat shock and transformants wereselected on yeast minimal medium (YMM) plates containing 2% raffinose asthe sole carbon source. Clonal inoculum cultures were established inliquid YMM with 2% raffinose as the sole carbon source. Experimentalcultures in were inoculated from these, in YMM+1% NP-40, to an initialOD₆₀₀ of ˜0.3. Cultures were grown at 30° C. with shaking (˜60 rpm)until OD₆₀₀ was approximately 1.0. At this point galactose was added toa final concentration of 2% and precursor fatty acids were added to afinal concentration of 0.5 mM. Cultures were incubated at 20° C. withshaking for a further 48 hours prior to harvesting by centrifugation.Cell pellets were washed with 1% NP-40, 0.5% NP-40 and water to removeany unincorporated fatty acids from the surface of the cells.

Gas Chromatography (GC) Analysis of Fatty Acids

Fatty Acid Preparation

Fatty acid methyl esters (FAME) were formed by transesterification ofthe centrifuged yeast pellet or Arabidopsis seeds by heating withMeOH—CHCl₃—HCl (10:1:1, v/v/v) at 90-100° C. for 2 h in a glass testtube fitted with a Teflon-lined screw-cap. FAME were extracted intohexane-dichloromethane (4:1, v/v) and analysed by GC and GC-MS.

Capillary Gas-Liquid Chromatography (GC)

FAME were analysed with Hewlett Packard (HP) 5890 GC or Agilent 6890 gaschromatograph fitted with HP 7673A or 6980 series automatic injectorsrespectively and a flame-ionization detector (FID). Injector anddetector temperatures were 290° C. and 310° C. respectively. FAMEsamples were injected at 50° C. onto a non-polar cross-linkedmethyl-silicone fused-silica capillary column (HP-5; 50 m×0.32 mm i.d.;0.17 μm film thickness.). After 1 min, the oven temperature was raisedto 210° C. at 30° C. min⁻¹, then to a final temperature of 280° C. at 3°C. min⁻¹ where it was kept for 5 min. Helium was the carrier gas with acolumn head pressure of 65 KPa and the purge opened 2 min afterinjection. Identification of peaks was based on comparison of relativeretention time data with standard FAME with confirmation usingmass-spectrometry. For quantification Empower software (Waters) orChemstation (Agilent) was used to integrate peak areas.

Gas Chromatography-Mass Spectrometry (GC-MS)

GC-MS was carried out on a Finnigan GCQ Plus GC-MS ion-trap fitted withon-column injection set at 4° C. Samples were injected using an AS2000auto sampler onto a retention gap attached to an HP-5 Ultra 2bonded-phase column (50 m×0.32 mm i.d.×0.17 μm film thickness). Theinitial temperature of 45° C. was held for 1 min, followed bytemperature programming at 30° C.·min⁻¹ to 140° C. then at 3° C.·min⁻¹to 310° C. where it was held for 12 min. Helium was used as the carriergas. Mass spectrometer operating conditions were: electron impact energy70 eV; emission current 250 μamp, transfer line 310° C.; sourcetemperature 240° C.; scan rate 0.8 scans·s⁻¹ and mass range 40-650Dalton. Mass spectra were acquired and processed with Xcalibur™software.

Construction of P. salina cDNA Library

mRNA, for the construction of a cDNA library, was isolated from P.salina cells using the following method. 2 g (wet weight) of P. salinacells were powdered using a mortar and pestle in liquid nitrogen andsprinkled slowly into a beaker containing 22 ml of extraction bufferthat was being stirred constantly. To this, 5% insolublepolyvinylpyrrolidone, 90 mM 2-mercaptoethanol, and 10 mM dithiotheitolwere added and the mixture stirred for a further 10 minutes prior tobeing transferred to a Corex™ tube. 18.4 ml of 3M ammonium acetate wasadded and mixed well. The sample was then centrifuged at 6000×g for 20minutes at 4° C. The supernatant was transferred to a new tube andnucleic acid precipitated by the addition of 0.1 volume of 3M NaAc (pH5.2) and 0.5 volume of cold isopropanol. After a 1 hour incubation at−20° C., the sample was centrifuged at 6000×g for 30 minutes in a swingrotor. The pellet was resuspended in 1 ml of water extracted withphenol/chloroform. The aqueous layer was transferred to a new tube andnucleic acids were precipitated once again by the addition of 0.1 volume3M NaAc (pH 5.2) and 2.5 volume of ice cold ethanol. The pellet wasresuspended in water, the concentration of nucleic acid determined andthen mRNA was isolated using the Oligotex mRNA system (Qiagen).

First strand cDNA was synthesised using an oligo-dT primer supplied withthe ZAP-cDNA synthesis kit (Stratagene—cat #200400) and the reversetranscriptase SuperscriptIII (Invitrogen). Double stranded cDNA wasligated to EcoRI/XhoI adaptors and from this a library was constructedusing the ZAP-cDNA synthesis kit as described in the accompanyinginstruction manual (Stratagene—cat #200400). The titer of the primarylibrary was 2.5×10⁵ plaque forming units (pfu)/ml and that of theamplified library was 2.5×10⁹ pfu/ml. The average insert size of cDNAinserts in the library was 1.3 kilobases and the percentage ofrecombinants in the library was 74%.

Example 2 Microalgae and Polyunsaturated Fatty Acid Contents Thereof

The CSIRO Collection of Living Microalgae

CSIRO established and maintained a Collection of Living Microalgae (CLM)containing over 800 strains from 140 genera representing the majority ofmarine and some freshwater microalgal classes (list of strains availabledownloadable from http://www.marine.csiro.au). Selectedmicro-heterotrophic strains were also maintained.

This collection is the largest and most diverse microalgal culturecollection in Australia. The CLM focused on isolates from Australianwaters—over 80% of the strains were isolated from diverse localities andclimatic zones, from tropical northern Australia to the AustralianAntarctic Territory, from oceanic, inshore coastal, estuarine,intertidal and freshwater environments. Additionally, emphasis has beenplaced on representation of different populations of a single species,usually by more than one strain. All strains in the culture collectionwere unialgal and the majority were clonal. A subset of strains wereaxenic. Another collection is the NIES-Collection (National Institutefor Environmental Studies, Environment Agency) maintained in Japan.

Microalgae are known for their cosmopolitanism at the morphologicalspecies level, with very low endemicity being shown. However thismorphological cosmopolitanism can hide a plethora of diversity at theintra-specific level. There have been a number of studies of geneticdiversity on different microalgae using approaches such asinterbreeding, isozymes, growth rates and a range of moleculartechniques. The diversity identified by these studies ranges from largeregional and global scales (Chinain et al., 1997) to between and withinpopulations (Gallagher, 1980; Medlin et al., 1996; Botch et al.,1999a,b). Variation at the intra-specific level, between morphologicallyindistinguishable microalgae, can usually only be identified usingstrains isolated from the environment and cultured in the laboratory.

It is essential to have identifiable and stable genotypes within culturecollections. While there are recorded instances of change or loss ofparticular characteristics in long term culture (Coleman, 1977), ingeneral, culturing guarantees genetic continuity and stability of aparticular strain. Cryopreservation strategies could also be used tolimit the potential for genetic drift.

Microalgae and their Use in Aquaculture

Because of their chemical/nutritional composition including PUFAs,microalgae are utilized in aquaculture as live feeds for various marineorganisms. Such microalgae must be of an appropriate size for ingestionand readily digested. They must have rapid growth rates, be amenable tomass culture, and also be stable in culture to fluctuations intemperature, light and nutrients as may occur in hatchery systems.Strains fulfilling these attributes and used widely in aquacultureinclude northern hemisphere strains such as Isochrysis sp. (T.ISO)CS-177, Pavlova lutheri CS-182, Chaetoceros calcitrans CS-178, C.muelleri CS-176, Skeletonema costarum CS-181, Thalassiosira pseudonanaCS-173, Tetraselmis suecica CS-187 and Nannochloropsis oculata CS-189.Australian strains used include Pavlova pinguis CS-375, Skeletonema sp.CS-252, Nannochloropsis sp. CS-246, Rhodomonas salina CS-24 and Naviculajeffreyi CS-46. Biochemical assessment of over 50 strains of microalgaeused (or of potential use) in aquaculture found that cells grown tolate-logarithmic growth phase typically contained 30 to 40% protein, 10to 20% lipid and 5 to 15% carbohydrate (Brown et al., 1997).

Lipid Composition Including PUFA Content of Microalgae

There is considerable interest in microalgae containing a high contentof the nutritionally important long-chain polyunsaturated fatty acids(LC-PUFA), in particular EPA [eicosapentaenoic acid, 20:5(ω3)] and DHA[docosahexaenoic acid, 22:6(ω3)] as these are essential for the healthof both humans and aquacultured animals. While these PUFA are availablein fish oils, microalgae are the primary producers of EPA and DHA.

The lipid composition of a range of microalgae (46 strains) andparticularly the proportion and content of important PUFA in the lipidof the microalgae were profiled. C₁₈-C₂₂ PUFA composition of microalgalstrains from different algal classes varied considerably across therange of classes of phototrophic algae (Table 3, FIG. 2, see alsoDunstan et. al., 1994, Volkman et al., 1989; Mansour et al., 1999a).Diatoms and eustigmatophytes were rich in EPA and produced small amountsof the less common PUFA, ARA [arachidonic acid, 20:4(ω6)] withnegligible amounts of DHA. In addition, diatoms made unusual C16 PUFAsuch as 16:4(ω1) and 16:3(ω4). In contrast, dinoflagellates had highconcentrations of DHA and moderate to high proportions of EPA andprecursor C₁₈ PUFA [18:5(ω3) and 18:4(ω3) SDA, stearidonic acid].Prymnesiophytes also contained EPA and DHA, with EPA the dominant PUFA.Cryptomonads were a rich source of the C₁₈ PUFA 18:3(ω3) (ALAα-linolenic acid) and SDA, as well as EPA and DHA. Green algae (e.g.Chlorophytes such as Dunaliella spp. and Chlorella spp.) were relativelydeficient in both C20 and C22 PUFA, although some species had smallamounts of EPA (up to 3%) and typically contained abundant ALA and18:2(ω6), and were also able to make 16:4(ω3). The biochemical ornutritional significance of uncommon C₁₆ PUFA [e.g. 16:4(ω3), 16:4(ω1),16:3(ω4)] and C₁₈ PUFA (e.g. 18:5(ω3) and STA] is unclear. However thereis current interest in C₁₈ PUFA such as SDA that are now beingincreasingly recognized as precursors for the beneficial EPA and DHA,unlike ALA which has only limited conversion to EPA and DHA.

New strains of Australian thraustochytrids were isolated. When examined,these thraustochytrids showed great morphological diversity from singlecells to clusters of cells, complex reticulate forms and motile stages.Thruastochytrids are a group of single cell organisms that produce bothhigh oil and LC-PUFA content. They were initially thought to beprimitive fungi, although more recently have been assigned to thesubclass Thraustochytridae (Chromista, Heterokonta), which aligns themmore closely with other heterokont algae (e.g. diatoms and brown algae).Under culture, thraustochytrids can achieve considerably higher biomassyield (>20 g/L) than other microalgae. In addition, thraustochytrids canbe grown in fermenters with an organic carbon source and thereforerepresent a highly attractive, renewable and contaminant-free, source ofomega-3 oils.

TABLE 3 Distribution of selected PUFA and LC-PUFA in microalgae andother groups, and areas of application. Group Genus/Species PUFAApplication Eustigmatophytes Nannochloropsis EPA Aquaculture DiatomsChaetoceros Dinoflagellates Crypthecodinium DHA Aquaculture, cohniihealth Thraustochytrids Schizochytrium supplements, infant formula Redalgae Phorphyridium ARA Aquaculture, infant formula Thraustochytridsundescribed species Pharmaceutical industry Fungi Mortiella (precursorto prostaglandins) Blue green algae Spirulina GLA health supplementsAbbreviations: γ-linolenic acid, GLA, 18:3ω6; 20:5ω3, eicosapentaenoicacid, EPA, 20:5ω3; docosahexaenoic acid, DHA, 22:6ω3; arachidonic acid,ARA, 20:4ω6.

Representative fatty acid profiles for selected Australianthraustochytrids are shown in Table 4. Strain O was particularlyattractive as it contained very high levels of DHA (61%). Other PUFAwere present at less than 5% each. High DHA-containing thraustochytridsoften also contained high proportions of 22:5ω6, docosapentaenoic acid(DPA), as was observed for strains A, C and H. DPA was only a minorcomponent in strain O under the culture conditions employed, making thisstrain particularly interesting. Strain A contained both DHA (28%) andEPA (16%) as the main LC-PUFA. Strains C and H differed from the otherstrains with ARA (10-13%) also being present as a major LC-PUFA. Anumber of other LC-PUFA were present in the thraustochytrids includingDPA(3) and 22:4ω6 and other components.

TABLE 4 Fatty acid composition (% of total) of thraustochytrid strains.Percentage composition Strain Fatty acid A C H O 16:0 18.0 16.4 13.522.1 20:4ω6 ARA 4.0 10.5 13.4 0.7 20:5ω3 EPA 15.8 7.7 5.2 4.1 22:5ω6DPA(6) 16.6 9.3 12.7 3.4 22:6ω3 DHA 28.2 21.6 19.2 61.0

The microalgal and thraustochytrid isolates in the CLM that may be usedfor isolation of genes involved in the synthesis of LC-PUFA are of thegenera or species as follows:

Class Bacillariophyceae (Diatoms)

Attheya septentrionalis, Aulacoseira sp., Chaetoceros affinis,Chaetoceros calcitrans, Chaetoceros calcitrans f. pumilum, Chaetoceroscf. mitra, Chaetoceros cf. peruvianus, Chaetoceros cf. radians,Chaetoceros didymus, Chaetoceros difficile, Chaetoceros gracilis,Chaetoceros muelleri, Chaetoceros simplex, Chaetoceros socialis,Chaetoceros sp., Chaetoceros cf. minus, Chaetoceros cf. tenuissimus,Coscinodiscus wailesii, other Coscinodiscus spp., Dactyliosolenfragilissimus, Detonula pumila, Ditylum brightwellii, Eucampia zodiacus,Extubocellulus spinifera, Lauderia annulata, Leptocylindrus danicus,Melosira moniliformis, Melosira sp., Minidiscus trioculatus,Minutocellus polymorphus, Odontella aurita, Odontella mobiliensis,Odontella regia, Odontella rhombus, Odontella sp., Papiliocellulussimplex, Planktosphaerium sp., Proboscia alata, Rhizosolenia imbricata,Rhizosolenia setigera, Rhizosolenia sp., Skeletonema costatum,Skeletonema pseudocostatum, Skeletonema sp., Skeletonema tropicum, otherSkeletonema spp., Stephanopyxis turris, Streptotheca sp., Streptothecatamesis, Streptotheca spp., Striatella sp., Thalassiosira delicatula,Thalassiosira eccentrica, Thalassiosira mediterranea, Thalassiosiraoceanica, Thalassiosira oestrupii, Thalassiosira profunda, Thalassiosirapseudonana, Thalassiosira rotula, Thalassiosira stellaris, otherThalassiosira spp., Achnanthes cf. amoena, Amphiprora cf. alata,Amphiprora hyalina, Amphora spp., Asterionella glacialis,Asterionellopsis glacialis, Biddulphia sp., Cocconeis sp., Cylindrothecaclosterium, Cylindrotheca fusiformis, Delphineis sp., Diploneis sp.,Entomoneis sp., Fallacia carpentariae, Grammatophora oceanica, Hasleaostrearia, Licmophora sp., Manguinea sp., Navicula cf. jeffreyi,Navicula jeffreyi, other Navicula spp., Nitzschia cf. bilobata,Nitzschia cf. constricta, Nitzschia cf. cylindrus, Nitzschia cf.frustulum, Nitzschia cf paleacea, Nitzschia closterium, Nitzschiafraudulenta, Nitzschia frustulum, Nitzschia sp., Phaeodactylumtricornutum, Pleurosigma delicatulum, other Pleurosigma spp.,Pseudonitzschia australis, Pseudonitzschia delicatissima,Pseudonitzschia fraudulenta, Pseudonitzschia pseudodelicatissima,Pseudonitzschia pungens, Pseudonitzschia sp., Pseudostaurosira shiloi,Thalassionema nitzschioides, or Thalassiothrix heteromorpha.Class ChrysophyceaeChrysolepidomonas cf. marina, Hibberdia spp., Ochromonas danica,Pelagococcus subviridis, Phaeoplaca spp., Synura shagnicola or otherChrysophyte spp.Class CryptophyceaeChroomonas placoidea, Chroomonas sp., Geminigera cryophila, Hemiselmissimplex, Hemiselmis sp., Rhodomonas baltica, Rhodomonas maculata,Rhodomonas salina, Rhodomonas sp. or other Cryptomonad spp.Class Dinophyceae (Dinoflagellates)Alexandrium affine, Alexandrium catenella, Alexandrium margalefi,Alexandrium minutum, Alexandrium protogonyaulax, Alexandrium tamarense,Amphidinium carterae, Amphidinium cf britannicum, Amphidinium klebsii,Amphidinium sp., Amphidinium steinii, Amylax tricantha,Cryptothecodinium cohnii, Ensiculifera sp., Fragilidium spp.,Gambierdiscus toxicus, Gymnodinium catenatum, Gymnodinium galathaneum,Gymnodinium galatheanum, Gymnodinium nolleri, Gymnodinium sanguineum, orother Gymnodinium spp., Gyrodinium pulchellum, or other Gyrodinium spp.,Heterocapsa niei, Heterocapsa rotundata, Katodinium cf. rotundatum,Kryptoperidinium foliaceum, Peridinium balticum, Prorocentrum gracile,Prorocentrum mexicanum, Prorocentrum micans, Protoceratium reticulatum,Pyrodinium bahamense, Scrippsiella cf. precaria, or other Scrippsiellaspp. Symbiodinium microadriaticum, or Woloszynskia sp.Class EuglenophyceaeEuglena gracilis.Class PrasinophyceaePycnococcus sp., Mantoniella squamata, Micrornonas pusilla, Nephroselmisminuta, Nephroselmis pynformes, Nephroselmis rotunda, Nephroselmis spp.,or other Prasinophyte spp., Pseudoscourfieldia marina, Pycnococcusprovasolii, Pyramimonas cordata, Pyramimonas gelidicola, Pyramimonasgrossii, Pyramimonas oltmansii, Pyramimonas propulsa, other Pyramimonasspp., Tetraselmis antarctica, Tetraselmis chuii, Tetraselmis sp.,Tetraselmis suecica, or other Tetraselmis spp.Class PrymnesiophyceaeChrysochromulina acantha, Chrysochromulina apheles, Chrysochromulinabrevifilum, Chrysochromulina camella, Chrysochromulina hirta,Chrysochromulina kappa, Chrysochromulina minor, Chrysochromulinapienaar, Chrysochromulina simplex, Chrysochromulina sp.,Chrysochromulina spinifera, Chrysochromulina strobilus, and otherChrysophyte spp., Chrysotila lamellosa, Cricosphaera carterae,Crystallolithus hyalinus, Diacronema vlkianum, Dicrateria inornata,Dicrateria sp., Emiliania huxleyi, Gephyrocapsa oceanica, Imantoniarotunda, and other Isochrysis spp., Ochrosphaera neapolitana, Pavlovacf. pinguis, Pavlova gyrans, Pavlova lutheri, Pavlova pinguis, Pavlovasalina, Pavlova sp., Phaeocystis cf. pouchetii, Phaeocystis globosa,Phaeocystis pouchetii, other Phaeocystis spp., Pleurochrysis aff.carterae, Prymnesium parvum, Prymnesium patelliferum, other Prymnesiumspp., or Pseudoisochrysis paradoxa.Class RaphidophyceaeChattonella antiqua, other Chattonella spp., Fibrocapsa japonica, otherFibrocapsa spp., Heterosigma akashiwo, Heterosigma carterae, or otherHeterosigma spp.Class ThraustochytridaeSchizochytrium spp., Thraustochytrium aureum, Thraustochytrium roseum,or other Thraustochytrium spp.Class Eustigmatophytae as a Source of Genes for EPA Production:Eustigmatos vischeri, Monodus subterraneus, Nannochlaropsis oculata,Nannochloropsis salina, Vischeria helvetica, Vischeria punctata,Chloridella neglecta, Chloridella simplex, Chlorobotrys regularis,Ellipsoidon parvum, Ellipsoidon solitare, Eustigmatos magnus,Eustigmatos polyphem, Goniochloris sculpta, Monodus subterraneus,Monodus unipapilla, Nannochloropsis gaditana, Nannochloropsis granulata,Nannochloropsis limnetica, Pseudocharaciopsis, ovalis,Pseudocharaciopsis texensis, Pseudostaurastrum limneticum, or Vischeriastellata

Example 3 Isolation of Zebrafish Δ5/6 Desaturase and FunctionalCharacterization in Yeast

As well as microalgae, some other organisms have the capacity tosynthesise LC-PUFA from precursors such as α-linolenic acid (18:3, ALA)(see FIG. 1) and some of the genes responsible for such synthesis havebeen isolated (see Sayanova and Napier, 2004). The genes involved inomega-3 C₂₀+PUFA biosynthesis have been cloned from various organismsincluding algae, fungi, mosses, plants, nematodes and mammals. Based onthe current understanding of genes involved in the synthesis of omega-3C₂₀+PUFA, synthesis of EPA in plants would require the transfer of genesencoding at least two desaturases and one PUFA elongase. The synthesisof DHA from EPA in plants would require the additional transfer of afurther desaturase and a further elongase (Sayanova and Napier, 2004).These enzymes are: for the synthesis of EPA, the sequential activitiesof a Δ6 desaturase, Δ6 elongase and a Δ5 desaturase is required. Basedon an alternative pathway operative in some algae, EPA may also besynthesised by the sequential activities of a Δ9 elongase, a Δ8desaturase and a Δ5 desaturase (Wallis and Browse, 1999; Qi et al.,2002). For the further conversion of EPA to DHA in plants, a furthertransfer of a Δ5 elongase and Δ4 desaturase will be required (Sayanovaand Napier, 2004).

Hastings et al. (2001) isolated a gene encoding a Δ5/Δ6 bifunctionaldesaturase from zebra fish (Dania rerio) and showed that, when expressedin yeast, the desaturase was able to catalyse the synthesis of both Δ6(GLA and SDA) and Δ5 (20:4 and EPA) fatty acids. The desaturase wastherefore able to act on both ω6 and ω3 substrates.

Isolation of the Zebrafish Δ5/Δ6 Desaturase

RNA was extracted using the RNAeasy system according to themanufacturers instructions (Qiagen) from freshly dissected zebrafishlivers. Based on the published sequence (Hastings et al. 2001), primers,sense, 5′-CCCAAGCTTACTATGGGTGGCGGAGGACAGC-3′ (SEQ ID NO:39) andantisense 5′-CCGCTGGAGTTATTTGTTGAGATACGC-3′ (SEQ ID NO:40) at the 5′ and3′ extremities of the zebrafish Δ5/6 ORF were designed and used in aone-step reverse transcription-PCR (RT-PCR, Promega) with the extractedRNA and using buffer conditions as recommended by the manufacturer. Asingle amplicon of size 1335 bp was obtained, ligated into pGEM-T easy(Promega) and the sequence confirmed as identical to that published.

A fragment containing the entire coding region (SEQ ID NO:38) wasexcised and ligated into the yeast shuttle vector pYES2 (Invitrogen).The vector pYES2 carried the URA3 gene, which allowed selection foryeast transformants based on uracil prototrophy. The inserted codingregion was under the control of the inducible GAL1 promoter andpolyadenylation signal of pYES2. The resultant plasmid was designatedpYES2-zfΔ5/6, for introduction and expression in yeast (Saccharomycescerevisiae).

Expression of Zebrafish Δ5/Δ6 Desaturase in Yeast

The gene construct pYES2-zfΔ5/6 was introduced into yeast strain S288.Yeast was a good host for analysing heterologous potential LC-PUFAbiosynthesis genes including desaturases and elongases for severalreasons. It was easily transformed. It synthesised no LC-PUFA of its ownand therefore any new PUFA made was easily detectable without anybackground problems. Furthermore, yeast cells readily incorporated fattyacids from growth media into cellular lipids, thereby allowing thepresentation of appropriate precursors to transformed cells containinggenes encoding new enzymes, allowing for confirmation of their enzymaticactivities.

Biochemical Analyses

Yeast cells transformed with pYES2-zfΔ5/6 were grown in YMM medium andinduced by the addition of galactose. The fatty acids 18:3ω3 (ALA, 0.5mM) or 20:4ω3 (ETA, 0.5 mM) were added to the medium as described above.After 48 hours incubation, the cells were harvested and fatty acidanalysis carried out by capillary gas-liquid chromatography (GC) asdescribed in Example 1. The analysis showed that 18:4ω3 (1.9% of totalfatty acid) was formed from 18:3ω3 and 20:5ω3 (0.24% of fatty acids)from 20:4ω3, demonstrating Δ6 desaturase activity and Δ5 desaturaseactivity, respectively. These data are summarized in Table 5 and confirmthe results of Hastings et al (2001).

Example 4 Isolation of C. elegans Elongase and FunctionalCharacterization in Yeast

Cloning of C. elegans Elongase Gene

Beaudoin and colleagues isolated a gene encoding an ELO-type fatty acidelongase from the nematode Caenorhabditis elegans (Beaudoin et al.,2000) and this gene was isolated as follows. Oligonucleotide primershaving the sequences 5′-GCGGGTACCATGGCTCAGCATCCGCTC-3′ (SEQ ID NO:41)(sense orientation) and 5′-GCGGGATCCTTAGTTGTTCTTCTTCTT-3′ (SEQ ID NO:42)(antisense orientation) were designed and synthesized, based on the 5′and 3′ ends of the elongase coding region. These primers were used in aPCR reaction to amplify the 867 basepair coding region from a C. elegansN2 mixed-stage gene library, using an annealing temperature of 58° C.and an extension time of 1 minute. The PCR amplification was carried outfor 30 cycles. The amplification product was inserted into the vectorpGEM™ T-easy (Promega) and the nucleotide sequence confirmed (SEQ IDNO:37). An EcoRI/BamHI fragment including the entire coding region wasexcised and inserted into the EcoRI/BglII sites of pSEC-TRP(Stratagene), generating pSEC-Ceelo, for introduction and expression inyeast. pSEC-TRP contains the TRP1 gene, which allowed for the selectionof transformants in yeast by tryptophan prototrophy, and the GAL1promoter for expression of the chimeric gene in an inducible fashion inthe presence of galactose in the growth medium.

TABLE 5 Enzymatic activities in yeast and Arabidopsis PrecursorSynthesised % Observed Clone PUFA PUFA (of total FA) activitypYES2-zfΔ5/6 18:3ω3 18:4ω3 1.9 Δ6 desaturase pYES2-zfΔ5/6 20:4ω3 20:5ω30.24 Δ5 desaturase pYES2zfΔ5/6, 18:3ω3 18:4ω3 0.82 Δ6 desaturasepSEC-Ceelo 20:3ω3 0.20 Δ9 elongase 20:4ω3 0.02 Δ6 elongase NOTpYES2-psΔ8 18:3ω3 18:4ω3 — Δ6 desaturase pYES2-psΔ8 20:3ω3 20:4ω3 0.12Δ8 desaturase pYES2-psELO1 18:2ω6 20:2ω6 — pYES2-psELO1 18:3ω3 —pYES2-psELO1 20:3ω3 22:3ω3 — pYES2-psELO1 20:4ω3 22:4ω3 — pYES2-psELO120:5ω3 22:5ω3 0.82 Δ5 elongase pYES-psELO2 18:2ω6 20:2ω6 0.12 Δ9elongase pYES-psELO2 18:3ω3 20:3ω3 0.20 Δ9 elongase pYES-psELO2 20:3ω322:3ω3 — pYES-psELO2 20:4ω3 22:4ω3 — pYES-psELO2 20:5ω3 22:5ω3 —Arabidopsis + — 18:3ω6 0.32 Δ5/6 desaturase, zfΔ5/6 & Ceelo — 18:4ω3 1.1Δ5/6/9 elongase (plant #1) — 20:4ω6 1.1 — 20:5ω3 2.1 — 20:3ω6 1.1 —20:4ω3 0.40 — 20:2ω6 3.2 — 20:3ω3 TR — 22:4ω6 0.06 — 22:5ω3 0.13 22:3ω60.03 TR, trace, not accurately determined.Functional Characterization of C. elegans Elongase Gene in Yeast

Yeast strain S288 was transformed, using the method described in Example1, with both vectors pYES2-zfΔ5/6 and pSEC-Ceelo simultaneously anddouble transformants were selected on YMM medium that lacked tryptophanand uracil. The transformants grew well on both minimal and enrichedmedia, in contrast to transformants of strain S288 carrying pSEC-Ceeloalone, in the absence of pYES2-zfΔ5/6, which grew quite poorly. Doubletransformants were grown in YMM medium and induced by the addition ofgalactose. The fatty acid 18:3ω3 (ALA, 0.5 mM) was added to the mediumand, after 48 hours incubation, cells were harvested and fatty acidanalysis carried out by capillary gas-liquid chromatography (GC) asdescribed in Example 1. The analysis showed that 18:4ω3 (0.82% of totalfatty acid) and 20:3ω3 (0.20%) were formed from 18:3ω3, and 20:4ω3(0.02% of fatty acids) from either of those, demonstrating the concertedaction of an elongase activity in addition to the Δ6 desaturase activityand Δ5 desaturase activity of the zebrafish desaturase (Table 5). Theconcerted action of a bifunctional Δ5/6 desaturase gene and an elongasegene has not been reported previously. In particular, the use of abifunctional enzyme, if showing the same activities in plant cells,would reduce the number of genes that would need to be introduced andexpressed. This also has not been reported previously.

Example 5 Coordinate Expression of Fatty Acid Desaturase and Elongase inPlants

Genetic Construct for Co-Expression of the Zebrafish Δ6/Δ5 Desaturaseand C. elegans Elongase in Plant Cells

Beaudoin and colleagues (2000) showed that the C. elegans Δ6 elongaseprotein, when expressed in yeast, could elongate the C18 Δ6 desaturatedfatty acids GLA and SDA, i.e. that it had Δ6 elongase activity on C18substrates. They also showed that the protein did not have Δ5 elongaseactivity on a C20 substrate in yeast. We tested, therefore, whether thiselongase would be able to elongate the Δ6 desaturated fatty acids GLAand SDA in Arabidopsis seed. Arabidopsis thaliana seed have been shownto contain both omega-6 (18:2, LA) and omega-3 (18:3, ALA) fatty acids(Singh et al, 2001). The presence of 18:3 in particular makesArabidopsis seed an excellent system to study the expression of genesthat could lead to the synthesis of omega-3 C₂₀+PUFA like EPA and DHA.

The test for elongase activity in Arabidopsis required the coordinateexpression of a Δ6 desaturase in the seed to first form GLA or SDA. Wechose to express the elongase gene in conjunction with the zebrafishdesaturase gene described above. There were no previous reports of theexpression of the zebra fish Δ6/Δ5 desaturase and C. elegans elongasegenes in plant cells, either individually or together.

Seed-specific co-expression of the zebra fish Δ6/Δ5 desaturase and C.elegans elongase genes was achieved by placing the genes independentlyunder the control of a −309 napin promoter fragment, designated Fp1(Stalberg et al., 1993). For plant transformation, the genes wereinserted into the binary vector pWvec8 that comprised an enhancedhygromycin resistance gene as selectable marker (Wang et al., 1997). Toachieve this, the C. elegans elongase coding region from Example 4 wasinserted as a blunt-end fragment between the Fp1 and Nos 3′polyadenylation/terminator fragment in the binary vector pWvec8, formingpCeloPWvec8. The zebrafish Δ5/Δ6 desaturase coding region from Example 3was initially inserted as a blunt end fragment between the Fp1 and Nos3′ terminator sequences and this expression cassette assembled betweenthe HindIII and ApaI cloning sites of the pBluescript cloning vector(Stratagene). Subsequently, the entire vector containing the desaturaseexpression cassette was inserted into the HindIII site of pCeloPWvec8,forming pZebdesatCeloPWvec8. The construct, shown schematically in FIG.3, was introduced into Agrobacterium strain AGLI (Valvekens et al.,1988) by electroporation prior to transformation into Arabidopsisthaliana, ecotype Columbia. The construct was also designated the “DO”construct, and plants obtained by transformation with this constructwere indicated by the prefix “DO”.

Plant Transformation and Analysis

Plant transformation was carried out using the floral dipping method(Clough and Bent, 1998). Seeds (T1 seeds) from the treated plants (T0plants) were plated out on hygromycin (20 mg/l) selective media andtransformed plants selected and transferred to soil to establish T1plants. One hygromycin resistant plant was recovered from a first screenand established in soil. The transformation experiment was repeated and24 further confirmed T1 transgenic plants were recovered and establishedin soil. Most of these T1 plants were expected to be heterozygous forthe introduced transgenes.

T2 seed from the 25 transgenic plants were collected at maturity andanalysed for fatty acid composition. As summarised in Table 6, seed ofuntransformed Arabidopsis (Columbia ecotype) contained significantamounts of both the ω6 and ω3, C18 fatty acid precursors LA and ALA butdid not contain any Δ6-desaturated C18 (18:36ω6 or 18:4ω3),ω6-desaturated C20 PUFA or ω3-desaturated C20 PUFA. In contrast, fattyacids of the seed oil of the transformed plants comprising the zebrafish Δ5/Δ6 desaturase and C. elegans elongase gene constructs contained18:3ω6, 18:4ω3 and a whole series of ω6-

TABLE 6 Fatty acid composition in transgenic seed (% of the total fattyacid in seed oil). Fatty acid Plant GLA SDA ARA EPA DGLA ETA EDA ETrADPA number 18:3ω6 18:4ω3 20:4ω6 20:5ω3 20:3ω6 20:4ω3 20:2ω6 20:3ω322:4ω6 22:5ω3 22:3ω6 Wt — — — — — — — — — — — DO1 0.32 1.10 1.10 2.101.10 0.40 3.20 TR 0.06 0.13 0.03 DO2 0.20 0.70 0.60 1.20 0.80 0.40 1.60— 0.10 TR — DO3 0.20 0.50 0.40 0.80 0.60 0.30 1.90 — TR TR — DO4 0.300.90 0.80 1.30 1.10 0.50 1.90 — — 0.10 — DO5 0.10 0.50 0.20 0.40 0.40 —0.30 — TR TR — DO6 0.30 1.00 1.00 1.70 1.20 0.50 2.50 — 0.10 0.10 — DO70.10 0.40 0.40 0.70 0.70 0.30 1.60 — TR TR — DO8 0.30 1.20 1.10 2.101.40 0.60 2.80 — 0.10 0.10 — DO9 0.30 1.30 0.90 2.20 1.30 0.60 3.10 —0.10 0.10 — DO10 0.10 0.40 0.30 0.70 0.50 0.30 0.10 — TR TR — DO11 0.301.00 1.40 2.30 1.50 0.60 3.20 — 0.10 0.20 — DO12 0.40 1.40 1.10 1.901.20 0.60 2.30 — 0.10 0.10 — DO13 0.20 0.60 0.60 0.90 0.80 0.40 0.40 —TR 0.10 — DO14 0.30 1.00 0.70 1.70 1.10 0.60 2.50 — TR TR — DO15 0.301.30 1.00 2.30 1.50 0.60 2.60 — 0.10 0.10 — DO17 0.20 0.40 0.40 0.700.70 0.30 1.80 — TR TR — DO18 0.20 0.60 0.50 0.90 0.80 0.40 1.70 — TR TR— DO19 0.20 0.40 0.40 0.80 0.70 0.30 2.00 — TR 0.10 DO20 0.30 1.00 0.500.90 0.70 0.30 1.60 — TR TR — DO21 0.30 1.20 0.90 2.00 1.30 0.60 2.50 —— 0.10 — DO22 0.30 0.90 0.70 1.20 1.00 0.40 0.30 — TR TR — DO23 — — — —0.10 0.10 1.80 — — — — DO24 0.30 1.10 0.70 1.50 1.10 0.50 2.90 — TR 0.10— DO25 0.10 0.50 0.30 0.70 0.50 0.20 1.60 — TR 0.10 — Wt = untransformedArabidopsis (Columbia). TR indicates less than 0.05%. Dash (—) indicatesnot detected.and ω3-C20 PUFA. These resulted from the sequential action of thedesaturase and elongase enzymes on the respective C18 precursors. Mostimportantly and unexpectedly, the transgenic seed contained both 20:5ω3(EPA), reaching at least 2.3% of the total fatty acid in the seedoil,and 22:5ω3 (DPA), reaching at least 0.2% of this omega-3 LC-PUFA in thefatty acid of the seedoil. The total C20 fatty acids produced in thetransgenic seed oil reached at least 9.0%. The total ω3 fatty acidsproduced that were a product of Δ6 desaturation (i.e. downstream of18:3ω3 (ALA), calculated as the sum of the percentages for 18:4ω3 (SDA),20:4ω3 (ETA), 20:5ω3 (EPA) and 22:5ω3 (DPA)) reached at least 4.2%.These levels represent a conversion efficiency of ALA, which is presentin seed oil of the wild-type Arabidopsis plants used for thetransformation at a level of about 13-15%, to ω3 products through a Δ6desaturation step of at least 28%. Stated otherwise, the ratio of ALAproducts to ALA (products:ALA) in the seed oil was at least 1:3.6. Ofsignificance here. Arabidopsis has a relatively low amount of ALA in itsseed oil compared to some commercial oilseed crops.

The T2 lines described above included lines that were homozygous for thetransgenes as well as heterozygotes. To distinguish homozygotes andheterozygotes for lines expressing the transgenes at the highest levels,T2 plants were established from the T2 seed for the 5 lines containingthe highest EPA levels, using selection on MS medium containinghygromycin (15 mg/L) to determine the presence of the transgenes. Forexample, the T2 seed was used from the T1 plant designated DO11,containing 2.3% EPA and showing a 3:1 segregation ratio of resistant tosusceptible progeny on the hygromycin medium, indicating that DO11contained the transgenes at a single genetic locus. Homozygous lineswere identified. For example, T2 progeny plant DO11-5 was homozygous asshown by the uniformly hygromycin resistance in its T3 progeny. Other T2plants were heterozygous for the hygromycin marker.

The fatty acid profiles of T3 seed lots from DO11-5 and other T2 progenyof DO11 were analysed and the data are presented in Table 7. Asexpected, the EPA contents reflected segregation of the DO construct.The levels of EPA in the fatty acid of the seedoil obtained from the T3lines were in three groups: negligible (nulls for the DO construct), inthe range 1.6-2.3% (heterozygotes for the DO construct) and reaching atleast 3.1% (homozygotes for the DO construct). The levels obtained werehigher in homozygotes than heterozygotes, indicating a gene dosageeffect. T3 seed from the DO11-5 plant synthesized a total of 9.6% new ω3and ω6 PUFAs, including 3.2% EPA, 1.6% ARA, 0.1% DPA, 0.6% SDA and 1.8%GLA (Table 7). This level of EPA synthesis in seed was four fold higherthan the 0.8% level previously achieved in linseed

TABLE 7 Fatty acid composition in transgenic seed (% of the total fattyacid in seed oil). Fatty acid Wildtype DO11-5 DO11-6 DO11-7 DO11-8DO11-10 DO11-11 DO11-12 14:0 0.3 0.0 0.1 0.1 0.1 0.1 0.0 0.1 15:0 0.00.0 0.2 0.2 0.2 0.1 0.0 0.1 16:1ω7 0.5 0.4 0.6 0.7 0.6 0.5 0.4 0.6 16:08.1 7.1 7.9 7.8 7.6 7.0 7.1 7.8 17:1ω8 0.0 0.0 0.0 0.1 0.1 0.1 0.0 0.117:0 0.3 0.1 0.0 0.1 0.1 0.1 0.0 0.1 18:3ω6 GLA 0.0 0.6 0.0 0.0 0.0 0.30.3 0.0 18:4ω3 SDA 0.0 1.8 0.0 0.0 0.0 1.0 1.1 0.0 18:2ω6 LA 26.6 25.829.8 28.6 28.8 25.6 25.4 28.6 18:1ω9 17.9 18.7 15.6 19.6 18.2 22.0 18.618.6 18:1ω7/ ALA 16.0 11.5 15.3 14.7 15.9 10.6 11.6 14.5 18:3ω3 18:0 3.44.2 2.9 2.7 2.8 3.5 3.9 2.8 19:0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 20:4ω6ARA 0.0 1.6 0.0 0.0 0.0 0.9 0.9 0.0 20:5ω3 EPA 0.0 3.2 0.0 0.1 0.0 1.62.1 0.0 20:3ω6 DGLA 0.0 1.9 0.0 0.0 0.0 1.2 1.5 0.0 20:4ω3 ETA 0.0 0.40.0 0.0 0.0 0.4 0.6 0.0 20:2ω6 0.0 3.4 0.2 0.1 0.2 2.2 3.1 0.1 20:1ω9/17.4 10.9 17.8 18.1 17.3 14.8 12.5 18.2 ω11 20:1ω7 1.9 2.7 2.2 1.9 2.22.2 2.3 2.0 20:0 1.8 1.8 2.1 1.8 2.0 2.0 2.0 2.0 22:4ω6 0.0 0.0 0.0 0.00.0 0.1 0.0 0.0 22:5ω3 DPA 0.0 0.1 0.0 0.0 0.0 0.1 0.1 0.0 22:1ω11/ 0.00.0 0.0 0.0 0.0 0.0 0.0 0.0 ω13 22:1ω9 1.3 0.8 1.9 1.7 1.7 1.5 1.1 1.722:1ω7 0.0 0.0 0.2 0.1 0.2 0.1 0.0 0.1 22:0 0.2 0.3 0.3 0.3 0.3 0.3 0.40.3 24:1ω9 0.6 0.4 0.2 0.2 0.3 0.2 0.2 0.2 24:1ω7 0.0 0.0 0.0 0.0 0.00.0 0.0 0.0 24:0 0.0 0.2 0.2 0.2 0.2 0.2 0.2 0.2 Fatty acid DO11-13DO11-16 DO11-18 DO11-19 DO11-20 DO11-21 14:0 0.1 0.1 0.1 0.1 0.1 0.115:0 0.1 0.1 0.1 0.1 0.2 0.1 16:1ω7 0.5 0.6 0.4 0.4 0.7 0.5 16:0 7.7 7.66.8 6.7 7.6 7.3 17:1ω8 0.1 0.0 0.1 0.1 0.0 0.1 17:0 0.1 0.1 0.1 0.1 0.00.1 18:3ω6 GLA 0.4 0.0 0.2 0.3 0.0 0.4 18:4ω3 SDA 1.3 0.0 0.7 1.1 0.01.2 18:2ω6 LA 25.6 29.0 25.7 25.2 29.4 27.3 18:1ω9 20.4 15.5 20.1 19.816.6 14.8 18:1ω7/ ALA 11.1 16.0 13.7 13.6 14.8 13.1 18:3ω3 18:0 3.9 2.93.3 3.4 2.9 3.7 19:0 0.1 0.0 0.1 0.1 0.0 0.1 20:4ω6 ARA 1.3 0.0 0.4 0.80.0 1.3 20:5ω3 EPA 2.1 0.0 1.1 1.8 0.0 2.3 20:3ω6 DGLA 1.4 0.0 0.7 1.00.0 1.5 20:4ω3 ETA 0.2 0.0 0.3 0.4 0.0 0.5 20:2ω6 2.4 0.2 1.7 2.1 0.12.8 20:1ω9/ 13.2 18.0 15.4 14.0 18.6 12.4 ω11 20:1ω7 2.0 2.3 2.2 2.2 2.32.7 20:0 1.9 2.2 2.0 2.0 2.3 2.1 22:4ω6 0.1 0.0 0.0 0.0 0.0 0.1 22:5ω3DPA 0.1 0.0 0.1 0.1 0.0 0.2 22:1ω11/ 0.0 0.0 0.0 0.0 0.0 0.0 ω13 22:1ω91.1 2.0 1.6 1.4 2.1 1.5 22:1ω7 0.0 0.2 0.1 0.1 0.2 0.2 22:0 0.3 0.4 0.30.3 0.4 0.4 24:1ω9 0.2 0.3 0.2 0.2 0.2 0.3 24:1ω7 0.0 0.0 0.0 0.0 0.00.0 24:0 0.2 0.2 0.2 0.2 0.2 0.3 Wild-type here refers to untransformedArabidopsis thaliana, ecotype Columbia(Abbadi et al., 2004). Considering also that the level of ALA precursorfor EPA synthesis in Arabidopsis seed was less than a third of thatpresent in linseed, it appeared that the LC-PUFA pathway as describedabove which included a desaturase that was capable of using an acyl-CoAsubstrate, was operating with significantly greater efficiency than theacyl-PC dependent desaturase pathway expressed in linseed.

The relative efficiencies of the individual enzymatic steps encoded bythe EPA construct can be assessed by examining the percentage conversionof substrate fatty acid to product fatty acids (including subsequentderivatives) in DO11-5. The zebra-fish Δ5/Δ6 desaturase exhibited strongΔ5 desaturation, with 89% of 20:4ω3 being converted to EPA and DPA, and45% of 20:3ω6 being converted to ARA, consistent with the previouslyreported preference of this enzyme for ω3 PUFA over ω6 PUFA substrates(Hastings et al., 2001). In comparison, Δ6-desaturation occurred atsignificantly lower levels, with 32% of ALA and 14% LA being convertedto Δ6-desaturated PUFA. Given that previous studies in yeast showed thisenzyme to actually have higher Δ6-desaturase activity than Δ5-desaturaseactivity, the lower Δ6-desaturation levels achieved in Arabidopsis seedscould be reflect a limited availability of ALA and LA substrates in theacyl-CoA pool (Singh et al., in press). The Δ6-elongase operated highlyefficiently, with 86% of GLA and 67% of SDA being elongated, suggestingthat this enzyme may have a slight preference for elongation of ω6-PUFAsubstrate.

The germination ability of the T2 (segregating) and T3 seed (homozygouspopulation) was assessed on MS medium and on soil. Seed from the EPA andDPA containing lines DO11 and DO11-5 showed the same timing andfrequency of germination as wild-type seed, and the T2 and T3 plants didnot have any apparent abnormal morphological features. Plant growthrates in vitro or in soil and the quantities of seed obtained from theplants were also unaffected. Including the germination of the T1 seedfrom which plant DO11 was obtained, the normal germination of seed ofthe DO11 line was thus observed over three generations. In addition,normal germination rates and timing were also observed for the other EPAand DPA containing seed. This feature was both important and notpredictable, as higher plants do not naturally produce EPA or DPA andtheir seed therefore has never previously contained these LC-PUFA.Germination requires the catabolism of stored seed oils and use forgrowth and as an energy supply. The observed normal germination ratesshowed that plant seed were able to carry out these processes using EPAand DPA, and that these compounds were not toxic.

It has been reported that a Δ4 desaturase encoded by a gene isolatedfrom Thraustochytrium spp and expressed in Brassica juncea leaves wasable to convert exogenously supplied DPA to DHA (Qiu et al., 2001). DPAproduced in the plant seed described herein can serve as a precursor forDHA production. This conversion of DPA to DHA may be achieved in plantcells by the introduction of a Δ4 desaturase gene into the DPA producingplant cells (Example 11).

Discussion

The presence of 22:5ω3 in the Arabidopsis seed oil implied that the C.elegans elongase gene not only had Δ6 elongase activity, but also Δ5elongase activity in plant cells. This result was most surprising giventhat the gene had been demonstrated to lack Δ5 elongase activity inyeast. Furthermore, this demonstrated that only two genes could be usedfor the synthesis of DPA from ALA in plant cells. The synthesis of DPAin a higher plant has not previously been reported. Furthermore, theconversion efficiency of ALA to its ω3 products in seed, including EPA.DPA or both, of at least 28% was striking.

Synthesis of LC-PUFA such as EPA and DHA in cells such as plant cells bythe Δ6 desaturation pathway required the sequential action of PUFAdesaturases and elongases. The required desaturases in one pathway hadΔ6, Δ5 and Δ4 desaturating activity, in that order, and the requiredPUFA elongases had elongating activity on Δ6 and Δ5 substrates. Thisconventional pathway operates in algae, mosses, fungi, diatoms,nematodes and some freshwater fish (Sayanova and Napier, 2004). The PUFAdesaturases from algae, fungi, mosses and worms are selective fordesaturation of fatty acids esterified to the sn-2 position ofphosphatidylcholine (PC) while the PUFA elongases act on fatty acids inthe form of acyl-CoA substrates represented in the acyl-CoA pool oftissues. In contrast, vertebrate Δ6 desaturases have been shown to beable to desaturate acyl-CoA substrates (Domergue et al., 2003a).

Attempts to reconstitute LC-PUFA pathways in plant cells and other cellshave to take into account the different sites of action and substraterequirements of the desaturases and elongase enzymes. For example, PUFAelongases are membrane bound, and perhaps even integral membraneproteins, which use acyl-CoAs which are present as a distinct pool inthe endoplasmic reticulum (ER). This acyl-CoA pool is physiologicallyseparated from the PC component of the ER, hence for a PUFA fatty acidto be sequentially desaturated and elongated it has to be transferredbetween PC and acyl-CoA pools in the ER. Therefore, earlier reportedattempts to constitute LC-PUFA biosynthesis in yeast using desaturasesand elongase from lower and higher plants, fungi and worms, have beeninefficient, at best. In addition, the constituted pathways have led tothe synthesis of only C20 PUFA such as ARA and EPA. There is no previousreport of the synthesis C22 PUFA such as DPA and DHA in yeast (Beaudoinet al., 2000, Domergue et al., 2003a).

The strategy described above of using a vertebrate desaturase, in thisexample a Δ5/Δ6 desaturase from zebra fish, with a Δ6 PUFA elongase fromC. elegans had the advantage that both the desaturase and the elongasehave activity on acyl-CoA substrates in the acyl-CoA pool. This mayexplain why this strategy was more efficient in the synthesis ofLC-PUFA. Furthermore, using a bifunctional desaturase displaying dualΔ5/Δ6 desaturase activities allowed the synthesis of EPA by the actionof only 2 genes instead of the 3 genes used by other researchers(Beaudoin et al., 2000, Domergue et al., 2003a). The use of abifunctional Δ5/Δ6 elongase in plant cells also allowed the formation ofDPA from ALA by the insertion of only three genes (one elongase and twodesaturases) or, as exemplified, of only two genes (bifunctionalelongase and bifunctional desaturase). Both of these aspects weresurprising and unexpected.

Biochemical evidence suggests that the fatty acid elongation consists of4 steps: condensation, reduction, dehydration and a second reduction.Two groups of condensing enzymes have been identified so far. The firstare involved in the synthesis of saturated and monosaturated fatty acids(C18-22). These are the FAE-like enzymes and do not appear to have arole in LC-PUFA biosynthesis. The other class of elongases identifiedbelong to the ELO family of elongases named after the ELO gene familywhose activities are required for the synthesis of the very long-chainfatty acids of sphingolipids in yeast. Apparent paralogs of the ELO-typeelongases isolated from LC-PUFA synthesizing organisms like algae,mosses, fungi and nematodes have been shown to be involved in theelongation and synthesis of LC-PUFA. It has been shown that only theexpression of the condensing component of the elongase is required forthe elongation of the respective acyl chain. Thus the introducedcondensing component of the elongase is able to successfully recruit thereduction and dehydration activities from the transgenic host to carryout successful acyl elongations. Thus far, successful elongations of C16and C18 PUFA have been demonstrated in yeast by the heterologousexpression of ELO type elongases. In this regard, the C. eleganselongase used as described above was unable to elongate C20 PUFA whenexpressed in yeast (Beaudoin et al, 2000). Our demonstration that the C.elegans elongase, when expressed in plants, was able to elongate theC20:5 fatty acid EPA as evidenced by the production of DPA inArabidopsis seed was a novel and unexpected result. One explanation asto why the C. elegans elongase was able to elongate C20 PUFA in plants,but not in yeast, might reside in its ability to interact successfullywith the other components of the elongation machinery of plants to bindand act on C20 substrates.

This example showed that an ELO-type elongase from a non-vertebrateorganism was able to elongate C20 PUFA in plant cells. Leonard et al.(2002) reported that an ELO-type elongase gene isolated from humans,when expressed in yeast, was able to elongate EPA to DPA but in anon-selective fashion.

Example 6 Isolation of a Δ8 Desaturase Gene from P. salina andFunctional Characterization in Yeast

Microalgae are the only organisms which have been reported to contain Δ8desaturases, aside from the Δ8 sphingolipid desaturases in higher plantsthat are not involved in LC-PUFA biosynthesis. A gene encoding a Δ8desaturase has been isolated from Euglena gracilis (Wallis and Browse,1999). The existence of a Δ5 desaturase in Isochrysis galbana may bepresumed because it contains a Δ9 elongase (Qi et al., 2002), theproduct of which, 20:3n-3, is the precursor for a Δ8 desaturase (seeFIG. 1). The fatty acid profiles of microalgae alone, however, do notprovide sufficient basis for identifying which microalgae will containΔ8 desaturase genes since multiple pathways may operate to produce theLC-PUFA.

Isolation of a Δ5 Desaturase Gene Fragment

An alignment of Δ6 desaturase amino acid sequences with those from thefollowing Genbank accession numbers, AF465283, AF007561, AAC15586identified the consensus amino acid sequence blocks DHPGGS (SEQ IDNO:43), WWKDKHN (SEQ ID NO:44) and QIEHHLF (SEQ ID NO:45) correspondingto amino acid positions 204-210 and 394-400, respectively, of AF465283.DHPGSS corresponded to the “cytochrome b5 domain” block that had beenidentified previously (Mitchell and Martin, 1995). WWKDKHN was aconsensus block that had not previously been identified or used todesign degenerate primers for the isolation of desaturase genes. TheQIEHHLF block, or variants thereof, corresponded to a requiredhistidine-containing motif that was conserved in desaturases. It hadbeen identified and used before as the “third His box” to designdegenerate oligonucleotides for desaturase gene isolation (Michaelson etal., 1998). This combination of blocks had not been used previously toisolate desaturase genes.

Based on the second and third conserved amino acid blocks, thedegenerate primers 5′-TGGTGGAARCAYAARCAYAAY-3′ (SEQ ID NO:46) and5′-GCGAGGGATCCAAGGRAANARRTGRTGYTC-3′ (SEQ ID NO:47) were synthesised.Genomic DNA from P. salina was isolated using the DNAeasy system(Qiagen). PCR amplifications were carried out in reaction volumes of 20μL using 20 pmol of each primer, 200 ng of P. salina genomic DNA andHotstar Taq DNA polymerase (Qiagen) with buffer and nucleotidecomponents as specified. The cycling conditions were: 1 cycle of 95° C.for 15 minutes; 5 cycles of 95° C. 1 min; 38° C., 1 min; 72° C., 1 min;followed by 35 cycles of 95° C., 35 sec; 52° C., 30 sec; 72° C., 1 min;and finishing with 1 cycle of 72° C., 10 min. A 515 basepair ampliconwas generated, ligated into pGEM-T easy (Promega), sequenced and used asa probe to screen a P. salina cDNA library.

Isolation of a cDNA Encoding a Δ8 Desaturase from P. salina

A P. salina cDNA library in λ-bacteriophage was constructed using theZap-cDNA Synthesis Kit (Stratagene) (see Example 1). The library wasplated out at a concentration of ˜50,000 plaques per plate and liftstaken with Hybond N+ membrane and treated using standard methods(Ausubel et al., 1988, supra). The 515 bp desaturase fragment, generatedby PCR, was radio-labelled with ³²P-dCTP and used to probe the liftsunder the following high stringency conditions: Overnight hybridisationat 65° C. in 6×SSC with shaking, a 5 minute wash with 2×SSC/0.1% SDSfollowed by two 10 minute washes with 0.2×SSC/0.1% SDS.

Fifteen primary library plates (150 mm) were screened for hybridizationto the labeled 515 bp fragment. Forty strongly hybridizing plaques wereidentified and ten of these were carried through to a secondary screen.Plasmids from five secondary plaques hybridizing to the 515 bp probewere excised with ExAssist Helper phage according to the suppliersprotocol (Stratagene). The nucleotide sequences of the inserts wereobtained using the ABI Prism Big Dye Terminator kit (PE AppliedBiosystems). The nucleotide sequences were identical where theyoverlapped, indicating that all five inserts were from the same gene.One of the five inserts was shown to contain the entire coding region,shown below to be from a Δ8 desaturase gene. This sequence is providedas SEQ ID NO:6.

The full-length amino acid sequence (SEQ ID NO:1) revealed that theisolated cDNA encoded a putative Δ6 or Δ8 desaturase, based on BLASTanalysis. These two types of desaturases are very similar at the aminoacid level and it was therefore not possible to predict on sequencealone which activity was encoded. The maximum degree of identity betweenthe P. salina desaturase and other desaturases (BLASTX) was 27-30%,while analysis using the GAP program which allows the insertions of“gaps” in the alignment showed that the maximum overall amino acididentity over the entire coding regions of the P. salina desaturase andAAD45877 from Euglena gracilis was 45%. A Pileup diagram of othersequences similar to the Pavlova salina desaturase is provided in FIG.4.

The entire coding region of this clone, contained within an EcoRI/XhoIfragment, was inserted into pYES2 (Invitrogen), generating pYES2-psΔ8,for introduction and functional characterisation in yeast. Cells ofyeast strain S288 were transformed with pYES2-psΔ8 as described inExample 1, and transformants were selected on medium without uracil. Theyeast cells containing pYES2-psΔ8 were grown in culture and then inducedby galactose. After the addition of 18:3ω3 or 20:3ω3 (0.5 mM) to theculture medium and 48 hours of further culturing at 30° C., the fattyacids in cellular lipids were analysed as described in Example 1. When18:3ω3 (Δ9, 12, 15) was added to the medium, no 18:4ω3 (Δ6, 9, 12, 15)was detected. However, when 20:3ω3 (Δ11,14,17) was added to the medium,the presence of 20:4ω3 (Δ8,11,14,17) in the cellular lipid of the yeasttransformants was detected (0.12%). It was concluded the transgeneencoded a polypeptide having Δ8 but not Δ6 desaturase activity in yeastcells.

Isolation of a gene encoding a Δ8 fatty acid desaturase that does notalso have Δ6 desaturase activity has not been reported previously. Theonly previously reported gene encoding a Δ8 desaturase that was isolated(from Euglena gracilis) was able to catalyse the desaturation of both18:3ω3 and 20:3ω3 (Wallis and Browse, 1999). Moreover, expression of agene encoding a Δ8 desaturase has not previously been reported in higherplants.

As shown in FIG. 1, expression of a Δ8 desaturase in concert with a Δ9elongase (for example the gene encoding ELO2—see below) and a Δ5desaturase (for example, the zebrafish Δ5/Δ6 gene or an equivalent genefrom P. salina or other microalgae) would cause the synthesis of EPA inplants.

Aside from providing an alternative route for the production of EPA incells, the strategy of using a Δ9 elongase in combination with the Δ8desaturase may provide an advantage in that the elongation, which occurson fatty acids coupled to CoA, precedes the desaturation, which occurson fatty acids coupled to PC, thereby ensuring the availability of thenewly elongated C20 PUFA on PC for subsequent desaturations by Δ8 and Δ5desaturases, leading possibly to a more efficient synthesis of EPA. Thatis, the order of reactions—an elongation followed by twodesaturations—will reduce the number of substrate linking switches thatneed to occur. The increased specificity provided by the P. salina Δ8desaturase is a further advantage.

Example 7 Isolation of P. salina ELO1 and ELO2 Fatty Acid Elongases

ELO-type PUFA elongases from organisms such as nematodes, fungi andmosses have been identified on the basis of EST or genome sequencingstrategies. A gene encoding a Δ9 elongase with activity on 18:30 (ALA)was isolated from Isochrysis galbana using a PCR approach withdegenerate primers, and shown to have activity in yeast cells that weresupplied with exogenous 18:2ω6 (LA) or 18:3ω3 (ALA), forming C20 fattyacids 20:2ω6 and 20:3ω3 respectively. The coding region of the geneIgASE1 encoded a protein of 263 amino acids with a predicted molecularweight of about 30 kDa and with limited homology (up to 27% identity) toother elongating proteins.

Isolation of Elongase Gene Fragments from P. salina

Based on multiple amino acid sequence alignments for fatty acidelongases the consensus amino acid blocks FLHXYH (SEQ ID NO:48) andMYXYYF (SEQ ID NO:49) were identified and the corresponding degenerateprimers 5′-CAGGATCCTTYYTNCATNNNTAYCA-3′ (SEQ ID NO:50) (sense) and5′-GATCTAGARAARTARTANNNRTACAT-3′ (SEQ ID NO:51) (antisense) weresynthesised. Primers designed to the motif FLHXYH or their use incombination with the MYXYYF primer have not previously been described.These primers were used in PCR amplification reactions in reactionvolumes of 20 μL with 20 pmol of each primer, 200 ng of P. salinagenomic DNA and Hotstar Taq DNA polymerase (Qiagen) with buffer andnucleotide components as specified by the supplier. The reactions werecycled as follows: 1 cycle of 95° C. for 15 minutes, 5 cycles of 95° C.,1 min, 38° C., 1 min, 72° C., 1 min, 35 cycles of 95° C., 35 sec, 52°C., 30 sec, 72° C., 1 min, 1 cycle of 72° C., 10 min. Fragments ofapproximately 150 bp were generated and ligated into pGEM-Teasy forsequence analysis.

Of the 35 clones isolated, two clones had nucleotide or amino acidsequence with similarity to known elongases. These were designated Elo1and Elo2. Both gene fragments were radio-labelled with ³²P-dCTP and usedto probe the P. salina cDNA library under the following high stringencyconditions: overnight hybridisation at 65° C. in 6×SSC with shaking, 5minute wash with 2×SSC/0.1% SDS followed by two 10 minute washes with0.2×SSC/0.1% SDS. Ten primary library plates (150 mm) were screenedusing the Elo1 or Elo2 probes. Elo1 hybridized strongly to severalplaques on each plate, whilst Elo2 hybridised to only three plaques inthe ten plates screened. All Elo1-hybridising plaques were picked from asingle plate and carried through to a secondary screen, whilst all threeElo2-hybridising plaques were carried through to a secondary screen.Each secondary plaque was then used as a PCR template using the forwardand reverse primers flanking the multiple cloning site in thepBluescript phagemid and the PCR products electrophoresed on a 1% TAEgel. Following electrophoresis, the gel was blotted onto a Hybond N+membrane and the membrane hybridised overnight with ³²P-labelled Elo1and Elo2 probes. Six of the amplified Elo1 secondary plaques and one ofthe amplified Elo2 secondary plaques hybridised to the Elo1/2 probe(FIG. 5).

Two classes of elongase-like sequences were identified in the P. salinacDNA library on the basis of their hybdridisation to the Elo1 and Elo2probes. Phagemids that hybridised strongly to either labelled fragmentwere excised with ExAssist Helper phage (Stratagene), and sequencedusing the ABI Prism Big Dye Terminator kit (PE Applied Biosystems). Allof the 5 inserts hybridizing to the Elo1 probe were shown to be from thesame gene. Similarly DNA sequencing of the 2 inserts hybridising to theElo2 probe showed them to be from the same gene. The cDNA sequence ofthe Elo1 clone is provided as SEQ ID NO:8, and the encoded protein asSEQ ID NO:2, whereas the cDNA sequence of the Elo2 clone is provided asSEQ ID NO:10, and the encoded proteins as SEQ ID NO:3, SEQ ID NO:85 andSEQ ID NO:86 using three possible start methionines).

A comparison was performed of the Elo1 and Elo2 and other known PUFAelongases from the database using the PILEUP software (NCBI), and isshown in FIG. 6.

The Elo1 cDNA was 1234 nucleotides long and had a open reading frameencoding a protein of 302 amino acid residues. According to the PILEUPanalysis, Elo1 clustered with other Elo-type sequences associated withthe elongation of PUFA including Δ6 desaturated fatty acids (FIG. 6).The Elo1 protein showed the greatest degree of identity (33%) to anelongase from the moss, P. patens (Accession No. AF428243) across theentire coding regions. The Elo1 protein also displayed a conserved aminoacids motifs found in all other Elo-type elongases.

The Elo2 cDNA was 1246 nucleotides long and had an open reading frameencoding a protein of 304 amino acid residues. According to PILEUPanalysis, Elo2 clustered with other Elo-type sequences associated withthe elongation of PUFA, including those with activity on Δ6 or Δ9 PUFA(FIG. 6). Elo2 was on the same sub-branch as the Δ9 elongase isolatedfrom Isochrysis galbana (AX571775). Elo2 displayed 31% identity to theIsochrysis gene across its entire coding region. The Elo2 ORF alsodisplayed a conserved amino acid motif found in all other Elo-typeelongases.

Example 8 Functional Characterization of Δ5 Fatty Acid Elongase in Yeastand Plant Cells

Yeast

The entire coding region of the P. salina Elo1 gene was ligated intopYES2, generating pYES2-psELO1, for characterisation in yeast. Thisgenetic construct was introduced into yeast strains and tested foractivity by growth in media containing exogenous fatty acids as listedin the Table 8. Yeast cells containing pYES2-psELO1 were able to convert20:50ω3 into 22:5ω3, confirming Δ5 elongase activity on C20 substrate.The conversion ratio of 7% indicated high activity for this substrate.The same yeast cells converted 18:4ω3 (Δ6,9,12,15) to 20:4ω3 and 18:3ω6(Δ6,9,12) to 20:3ω6, demonstrating that the elongase also had Δ6elongase activity in yeast cells, but at approximately 10-fold lowerconversion rates (Table 8). This indicated that the Elo1 gene encodes aspecific or selective Δ5 elongase in yeast cells. This represents thefirst report of a specific Δ5 elongase, namely an enzyme that has agreater Δ5 elongase activity when compared to Δ6 elongase activity. Thismolecule is also the first Δ5 elongase isolated from an algael source.This enzyme is critical in the conversion of EPA to DPA (FIG. 1).

Plants

The Δ5 elongase, Elo1 isolated from Pavlova is expressed in plants toconfirm its ability to function in plants. Firstly, a plant expressionconstruct is made for constitutive expression of Elo1. For this purpose,the Elo1 sequence is placed under the control of the 35S promoter in theplant binary vector pBI121 (Clontech). This construct is introduced intoArabidopsis using the floral dip method described above. Analysis ofleaf lipids is used to determine the specificity of fatty acidselongated by the Elo1 sequence. In another approach, co expression ofthe Elo1 construct with the zebra fish Δ5/Δ6 desaturase/C. eleganselongase construct and the Δ4 desaturase isolated from Pavlova, resultsin DHA synthesis from ALA in Arabidopsis seed, demonstrating the use ofthe Δ5 elongase in producing DHA in cells. In a further approach, theElo1 gene may be co-expressed with Δ6-desaturase and Δ5 desaturasegenes, or a Δ6/Δ5 bifunctional desaturase gene, to produce DPA from ALAin cells, particularly plant cells. In an alternative approach, the Δ5elongase and Δ4 elongase genes are used in combination with the PKSgenes of Shewanella which produce EPA (Takeyama et al., 1997), inplants, for the synthesis of DHA.

TABLE 8 Conversion of fatty acids in yeast cells transformed withgenetic constructs expressing Elo1 or Elo2. Fatty acid precursor/ Fattyacid formed/ Conversion ratio Clone (% of total FA) (% of total FA) (%)pYES2- 20:5n-3/3% 22:5n-3/0.21%   7% psELO1 pYES2- 18:4n-3/16.9%20:4n-3/0.15% 0.89% psELO1 pYES2- 18:3n-6/19.8% 20:3n-6/0.14% 0.71%psELO1 pYES2- 20:5n-3/2.3% 22:5n-3/tr — psELO2 pYES2- 18:4n-3/32.5%20:4n-3/0.38%  1.2% psELO2 pYES2- 18:3n-6/12.9% 20:3n-6/0.08% 0.62%psELO2 pYES2- 18:2n-6/30.3% 20:2n-6/0.12% 0.40% psELO2 pYES2-18:3n-3/42.9% 18:3n-3/0.20% 0.47% psELO2 tr: trace amounts (<0.02%)detected.

Example 9 Functional Characterization of Δ9 Fatty Acid Elongase in Yeastand Plant Cells

Expression in Yeast Cells

The entire coding region of the P. salina Elo2 gene encoding a proteinof 304 amino acids (SEQ ID NO:3) was ligated into pYES2, generatingpYES2-psELO2, for characterisation in yeast. This genetic construct wasintroduced into yeast strains and tested for activity by growth in mediacontaining exogenous fatty acids. Yeast cells containing pYES2-psELO2were able to convert 18:2ω6 into 20:2ω6 (0.12% of total fatty acids) and18:3ω3 into 20:3ω3 (0.20%), confirming Δ9 elongase activity on C18substrates (Table 8). These cells were also able to convert 18:3ω6 into20:3ω6 and 18:4ω3 into 20:4ω3, confirming Δ6 elongase activity on C18substrates in yeast. However, since the 18:3ω6 and 18:4ω3 substratesalso have a desaturation in the Δ9 position, it could be that the Elo2enzyme is specific for Δ9-desaturated fatty acids, irrespective ofwhether they have a Δ6 desaturation as well. The cells were able toconvert 20:5ω3 into the 22:5 product DPA. This is the first report of aΔ9 elongase that also has Δ6 elongase activity from a non-vertebratesource, in particular from a fungal or algal source.

As the coding region contained three possible ATG start codonscorresponding to methionine (Met) amino acids at positions 1, 11 (SEQ IDNO:85) and 29 (SEQ ID NO:86) of SEQ ID NO:3, the possibility thatpolypeptides beginning at amino acid positions 11 or 29 would also beactive was tested. Using 5′ oligonucleotide (sense) primerscorresponding to the nucleotide sequences of these regions, PCRamplification of the coding regions was performed, and the resultantproducts digested with EcoRI. The fragments are cloned into pYES2 toform pYES2-psELO2-11 and pYES2-psELO2-29. Both plasmids are shown toencode active Δ9-elongase enzymes in yeast. The three polypeptides mayalso be expressed in Synechococcus or other cells such as plant cells todemonstrate activity.

Expression in Plant Cells

The Δ9 elongase gene, Elo2, isolated from Pavlova was expressed inplants to confirm its ability to function in plants. Firstly, a plantexpression construct is made for constitutive expression of Elo2. Forthis purpose, the Elo2 coding sequence from amino acid position 1 of SEQID NO:3 was placed under the control of the 35S promoter in the plantbinary vector pBI121 (Clontech). This construct is introduced intoArabidopsis using the floral dip method described above. Analysis ofleaf lipids indicates the specificity of fatty acids that are elongatedby the Elo2 sequence.

Co-Expression of Δ9 Elongase and Δ8-Desaturase Genes in TransformedCells

The P. salina Δ8-desaturase and Δ9-elongase were cloned into a singlebinary vector, each under the control of the constitutive 35S promoterand nos terminator. In this gene construct, pBI121 containing theΔ8-desaturase sequence was cut with HindIII and ClaI (blunt-ended) torelease a fragment containing the 35S promoter and the Δ8-desaturasegene, which was then ligated to the HindIII+Sad (blunt ended) cutpXZP143/Δ9-elongase vector to result in the intermediate pJRP013. Thisintermediate was then opened with HindIII and ligated to apWvec8/Δ9-elongase binary vector (also HindIII-opened) to result in theconstruct pJRP014, which contains both genes between the left and rightT-DNA borders, together with a hygromycin selectable marker genesuitable for plant transformation.

This double-gene construct was then used to transform tobacco using astandard Agrobacterium-mediated transformation technique. Followingintroduction of the construct into Agrobacterium strain AGLI, a singletransformed colony was used to inoculate 20 mL of LB media and incubatedwith shaking for 48 hours at 28° C. The cells were pelleted (1000 g for10 minutes), the supernatant discarded, and the pellet resuspended in 20mL of sterile MS media. This was step was then repeated before 10 ml ofthis Agrobacterial solution was added to freshly cut (1 cm squares)tobacco leaves from cultivar W38. After gentle mixing, the tobacco leafpieces and Agrobacterium solution were allowed to stand at roomtemperature for 10 min. The leaf pieces were transferred to MS plates,sealed, and incubated (co-cultivation) for 2 days at 24° C. Transformedcells were selected on medium containing hygromycin, and shootsregenerated. These shoots were then cut off and transferred toMS-rooting media pots for root growth, and eventually transferred tosoil. Both leaf and seed lipids from these plants are analysed for thepresence of 20:2ω6, 20:3ω6, 20:3ω3 and 20:4ω3 fatty acids, demonstratingthe co-expression of the two genes.

Discussion

Biochemical evidence suggests that the fatty acid elongation consists of4 steps: condensation, reduction, dehydration and a second reduction,and the reaction is catalysed by a complex of four proteins, the firstof which catalyses the condensation step and is commonly called theelongase. There are 2 groups of condensing enzymes identified so far.The first are involved in the synthesis of saturated and monounsaturatedfatty acids (C18-22). These are the FAE-like enzymes and do not play anyrole in LC-PUFA biosynthesis. The other class of elongases identifiedbelong to the ELO family of elongases named after the ELO gene familywhose activities are required for the synthesis of very LC fatty acidsof sphingolipids in yeast. Apparent paralogs of the ELO-type elongasesisolated from LC-PUFA synthesizing organisms like algae, mosses, fungiand nematodes have been shown to be involved in the elongation andsynthesis of LC-PUFA. It has been shown that only the expression of thecondensing component of the elongase is required for the elongation ofthe respective acyl chain. Thus the introduced condensing component ofthe elongase is able to successfully recruit the reduction anddehydration activities from the transgenic host to carry out successfulacyl elongations. This was also true for the P. saliva Δ9-elongase.

Example 10 Isolation of a Gene Encoding a Δ4-Desaturase from P. salina

The final step in the aerobic pathway of DHA synthesis in organismsother than vertebrates, such as microorganisms, lower plants includingalgae, mosses, fungi, and possibly lower animals, is catalysed by aΔ4-desaturase that introduces a double bond into the carbon chain of thefatty acid at the Δ4 position. Genes encoding such an enzyme have beenisolated from the algae Euglena and Pavlova and from Thraustochytrium,using different approaches. For example, Δ4-desaturase genes fromPavlova lutheri and Euglena gracilis were isolated by random sequencingof cloned ESTs (EST approach, Meyer et al., 2003; Tonon et al., 2003),and a Δ4-desaturase gene from Thraustochytrium sp. ATCC21685 wasisolated by RT-PCR using primers corresponding to a cytochrome b₅ HPGGdomain and histidine box III region (Qiu et al., 2001). The clonedΔ4-desaturase genes encoded front-end desaturases whose members arecharacterised by the presence of an N-terminal cytochrome b₅-like domain(Napier et al., 1999; Sayanova and Napier, 2004).

Isolation of a Gene Fragment from a Δ4-Desaturase Gene from P. salina

Comparison of known moss and microalgae Δ4-desaturases revealed severalconserved motifs including a HPGG (SEQ ID NO:52) motif within acytochrome b₅-like domain and three histidine box motifs that arepresumed to be required for activity. Novel degenerate PCR primersPavD4Des-F3 (5′-AGCACGACGSSARCCACGGCG-3′) (SEQ ID NO:53) and PavD4Des-R3(5′-GTGGTGCAYCABCACGTGCT-3′) (SEQ ID NO:54) corresponding to theconserved amino acid sequence of histidine box I and complementary to anucleotide sequence encoding the amino acid sequence of histidine boxII, respectively, were designed as to amplify the corresponding regionof P. salina desaturase genes, particularly a Δ4-desaturase gene. Theuse of degenerate PCR primers corresponding to histidine box I andhistidine box II regions of Δ4-desaturase has not been reportedpreviously.

PCR amplification reactions using these primers were carried out usingP. salina first strand cDNA as template with cycling of 95° C., 5 minfor 1 cycle, 94° C. 30 sec, 57° C. 30 sec, 72° C. 30 sec for 35 cycles,and 72° C. 5 min for 1 cycles. The PCR products were cloned intopGEM-T-easy (Promega) vectors, and nucleotide sequences were determinedwith an ABI3730 automatic sequencer using a reverse primer from thepGEM-Teasy vector. Among 14 clones sequenced, three clones showedhomology to Δ4-desaturase genes. Two of these three clones are truncatedat one primer end. The nucleotide sequence of the cDNA insert of thethird, clone 1803, is provided as SEQ ID NO:11.

The amino acid sequence encoded by SEQ ID NO:11 was used to search theNCBI protein sequence database using the BLASTX software. The resultsindicated that this sequence was homologous to known Δ4-desaturases. Theamino acid sequence of the P. salina gene fragment showed 65%, 49%, 46%and 46% identity to that of Δ4-desaturases of P. lutheri,Thraustochytrium sp. ATCC21685. Thraustochytrium aureum and Euglenagracilis respectively.

Isolation of a Full-Length Δ4-Desaturase Gene

The insert from clone 1803 was excised, and used as probe to isolatefull-length cDNAs corresponding to the putative Δ4-desaturase genefragment. About 750,000 pfu of the P. salina cDNA library were screenedat high stringency. The hybridization was performed at 60° C. overnightand washing was done with 2×SSC/0.1% SDS 30 min at 65° C. then with0.2×SSC/0.1% SDS 30 min at 65° C. Eighteen hybridising clones wereisolated and secondary screening with six clones was performed under thesame hybridization conditions. Single plaques from secondary screeningof these six clones were isolated. Plasmids from five single plaqueswere excised and the nucleotide sequences of the inserts determined withan ABI 3730 automatic sequencer with reverse and forward primers fromthe vector. Sequencing results showed that four clones each containedΔ4-desaturase cDNA of approximately 1.7 kb in length, each with the samecoding sequence and each apparently full-length. They differed slightlyin the length of the 5′ and 3′ UTRs even though they contained identicalprotein coding regions. The cDNA sequence of the longest P. salinaM-desaturase cDNA is provided as SEQ ID NO:13, and the encoded proteinas SEQ ID NO:4.

The full-length cDNA was 1687 nucleotides long and had a coding regionencoding 447 amino acids. The Pavlova salina Δ4-desaturase showed allthe conserved motifs typical of ‘front-end desaturases’ including theN-terminal cytochrome b₅-like domain and three conserved histidine-richmotifs. Comparison of the nucleotide and amino acid sequences with otherΔ4-desaturase genes showed that the greatest extent of homology was forthe P. lutheri Δ4-desaturase (Accession No. AY332747), which was 69.4%identical in nucleotide sequence over the protein coding region, and67.2% identical in amino acid sequence.

Demonstration of Enzyme Activity of Pavlova salina Δ4-Desaturase Gene

A DNA fragment including the Pavlova salina Δ4-desaturase cDNA codingregion was excised as an EcoRI-SalI cDNA fragment and inserted into thepYES2 yeast expression vector using the EcoRI and XhoI sites. Theresulted plasmid was transformed into yeast cells. The transformantswere grown in YMM medium and the gene induced by the addition ofgalactose, in the presence of added (exogenous) ω6 and ω3 fatty acids inorder to demonstrate enzyme activity and the range of substrates thatcould be acted upon by the expressed gene. The fatty acids 22:5ω3 (DPA,1.0 mM), 20:4n-3 (ETA, 1.0 mM), 22:4ω6 (DTAG, 1.0 mM) and 20:4ω6 (ARA,1.0 mM) were each added separately to the medium. After 72 hoursincubation, the cells were harvested and fatty acid analysis carried outby capillary gas-liquid chromatography (GC) as described in Example 1.The data obtained are shown in Table 9.

TABLE 9 Yeast PUFA feeding showing activity of delta-4 desaturase gene.Exogenous fatty acid added Fatty acid composition to growth medium (% oftotal fatty acid) 22:4ω6 22:5ω3 14:0 0.63 0.35 15:0 0.06 0.06 16:1ω7c43.45 40.52 16:1ω5 0.20 0.13 16:0 18.06 15.42 17:1ω8 0.08 0.09 17:0 0.08— 18:1ω9 26.73 30.07 18:1ω7 (major) & 1.43 1.61 18:3ω3 18:1ω5c 0.02 tr18:0 7.25 8.87 20:5ω3 0.40 0.62 20:1ω9/ω11 0.03 tr 20:0 0.08 0.09 22:5ω60.03 0.00 22:6ω3 — 0.04 22:4ω6 0.97 — 22:5ω3 0.00 1.66 22:0 0.06 0.0624:1ω7 0.31 0.37 24:0 0.12 0.04 Sum 100.00% 100.00%

This showed that the cloned gene encoded a Δ4-desaturase which was ableto desaturate both C22:4ω6 (3.0% conversion to 22:5ω6) and C22:5ω3 (2.4%conversion to 22:6ω3) at the M position. The enzyme did not show any Δ5desaturation activity when the yeast transformants were fed C20:3ω6 orC20:4ω3.

Example 11 Expression of P. salina Δ4-Desaturase Gene in Plant Cells andProduction of DHA

To demonstrate activity of the Δ4-desaturase gene in plant cells, thecoding region may be expressed either separately to allow the conversionof DPA to DHA, or in the context of other LC-PUFA synthesis genes suchas, for example, a Δ5-elongase gene for the conversion of EPA to DHA.For expression as a separate gene, the Δ4-desaturase coding region maybe excised as a BamHI-SalI fragment and inserted between a seed-specificpromoter and a polyadenylation/transcription termination sequence, suchas, for example, in vector pGNAP (Lee et al., 1998), so that it isexpressed under the control of the seed specific promoter. Theexpression cassette may then be inserted into a binary vector andintroduced into plant cells. The plant material used for thetransformation may be either untransformed plants or transformed plantscontaining a construct which expressed the zebrafish Δ5/Δ6-dualdesaturase gene and C. elegans elongase gene each under the control of aseed specific promoter (Example 5). Transgenic Arabidopsis containingthe latter, dual-gene construct had successfully produced EPA and DPA inseeds, and the combination with the Δ4-desaturase gene would allow theconversion of the DPA to DHA in the plant cells, as demonstrated below.

To demonstrate co-expression of a Δ5-elongase gene with theΔ4-desaturase gene in recombinant cells, particularly plant cells, andallow the production of DHA, the Δ4-desaturase and the Δ5-elongase genesfrom P. salina (Example 8) were combined in a binary vector as follows.Both coding regions were placed under the control of seed-specific(napin) promoters and nos3′ terminators, and the binary vector constructhad a kanamycin resistance gene as a selectable marker for selection inplant cells. The coding region of the Δ5-elongase gene was excised fromits cDNA clone as a PstI-SacII fragment and inserted into anintermediate plasmid (pXZP143) between the promoter and terminator,resulting in plasmid pXZP144. The coding region of the Δ4-desaturasegene was excised from its cDNA clone as a BamHI-SalI fragment andinserted into plasmid pXZP143 between the promoter and nos 3′transcription terminator, resulting in plasmid pXZP150. These twoexpression cassettes were combined in one vector by inserting theHindIII-ApaI fragment from pXZP144 (containing promoter-Elo1-nos3′)between the StuI and ApaI sites of pXZP150, resulting in plasmidpXZP191. The HindIII-StuI fragment from pXZP191 containing bothexpression cassettes was then cloned into the binary vector pXZP330, aderivative of pBI121, resulting in plant expression vector pXZP355. Thisvector is shown schematically in FIG. 7.

Plant Transformation

The Δ5-elongase and the Δ4-desaturase genes on pXZP355 were introducedby the Agrobacterium-mediated floral dip transformation method into theArabidopsis plants designated DO11 (Example 5) which were alreadytransgenic for the zebrafish Δ5/Δ6 bifunctional desaturase and the C.elegans Δ5/Δ6 bifunctional elongase genes. Since those transgenes werelinked to a hygromycin resistance gene as a selectable marker gene, thesecondary transformation with pXZP355 used a kanamycin resistanceselection, thus distinguishing the two sets of transgenes. Fivetransgenic plants are obtained, designated “DW” plants. Since the DO11plants were segregating for the zebrafish Δ5/Δ6 bifunctional desaturaseand the C. elegans Δ5/Δ6 bifunctional elongase genes, some of thetransformed plants were expected to be heterozygous for these genes,while others were expected to be homozygous. Seed (T2 seed) of the fivetransformed plants were analysed and shown to contain up to at least0.1% DPA and up to at least 0.5% DHA in the seed oils. Data arepresented for two lines in Table 10. Analysis, by mass spectrometry(GC-MS), of the fatty acids in the peaks identified as EPA and DHA fromthe GC analysis proved that they were indeed EPA and DHA (FIG. 8).

The fatty acid analysis of the T2 seedoil demonstrated that significantconversion of EPA to DHA had occurred in the DW2 and DW5 lines, having0.2% and 0.5% DHA, respectively. Examination of the enzyme efficienciesin plant DW5 containing the higher level of DHA showed that 17% of theEPA produced in its seed was elongated to DPA by the P. salinaΔ5-elongase, and greater than 80% of this DPA was converted to DHA bythe P. salina Δ4-desaturase. Since the Δ5-elongase and Δ4-desaturasegenes were segregating in the T2 seed, the fatty acid composition datarepresented an average of pooled null, heterozygous and homozygousgenotypes for these genes. It is expected that levels of DHA in progenylines of DW5 will be greater in seed that is uniformly homozygous forthese genes.

TABLE 10 Fatty acid composition (% of total fatty acids) of seed oilsfrom Arabidopsis thaliana (ecotype Columbia) and derivatives carryingEPA and DHA gene constructs - EPA, DPA and DHA synthesis in transgenicseed. DO11 + DHA construct Wild type DW2 DW5 Fatty acid Columbia TotalTotal TAG PL Usual fatty acids 16:0 7.2 6.7 6.1 5.5 12.5 18:0 2.9 3.84.4 4.3 4.5 18:1Δ⁹ 20.0 20.6 16.6 18.9 13.7 18:2Δ^(9,12) (LA) 27.5 26.025.9 25.5 33.1 18:3Δ^(9,12,15) (ALA) 15.1 13.2 15.0 13.6 15.1 20.0 2.22.1 1.8 1.9 0.6 20:1Δ¹¹ 19.8 14.8 10.5 10.5 3.2 20:1Δ¹³ 2.2 3.0 4.2 4.81.4 20:2Δ^(11,14) 0.1 1.7 3.5 3.8 3.7 22:1Δ¹³ 1.5 1.4 1.0 0.3 0.4 Otherminor 1.5 2.9 2.7 2.4 3.8 Total 100.0 96.0 91.7 91.5 92.0 New ω6-PUFA18:3Δ^(6,9,12) (GLA) 0 0.2 0.4 0.4 0.2 20:3Δ^(8,11,14) 0 0.8 1.5 1.5 1.720:4Δ^(5,8,11,14) (ARA) 0 0.4 1.0 1.1 1.2 22:4Δ^(7,10,13,16) 0 0 0 0 0.222:5Δ^(4,7,10,13,16) 0 0 0.1 0.1 0.1 Total 0 1.4 3.0 3.1 3.4 New ω3-PUFA18:4Δ^(6,9,12,15) (SDA) 0 0.7 1.5 1.6 0.5 20:4Δ^(8,11,14,17) 0 0.5 0.80.7 0.9 20:5Δ^(5,8,11,14,17) (EPA) 0 1.1 2.4 2.5 2.322:5Δ^(7,10,13,16,19) (DPA) 0 0.1 0.1 0.2 0.7 22:6Δ^(4,7,10,13,16,19)(DHA) 0 0.2 0.5 0.4 0.2 Total 0 2.6 5.3 5.4 4.6 Total fatty acids 100.0100.0 100.0 100.0 100.0 Total MUFA^(a) 41.3 36.8 28.1 29.7 17.3 TotalC₁₈-PUFA^(b) 42.6 39.2 40.9 39.1 48.2 Total new PUFA^(c) 0 4.0 8.3 8.58.0 ^(a)Total of 18:1Δ⁹ and derived LC-MUFA (=18:1Δ⁹ + 20:1Δ¹¹ +22:1Δ¹³) ^(b)18:2 + 18:3 ^(c)Total of all new ω6 and ω3-PUFA

Germination of 50 T2 seed from each of DW2 and DW5 onhygromycin-containing medium showed that the DW5 T1 plant was homozygous(50/50) for the Δ5/Δ6 bifunctional desaturase and Δ5/Δ6 bifunctionalelongase genes, while the DW2 seed segregating in a 3:1 ration(resistant: susceptible) for these genes and DW2 was thereforeheterozygous. This was consistent with the higher levels of EPA observedin DW5 seed compared to DW2 seed, and explained the increased level ofDHA produced in the seed homozygous for these transgenes. This furtherdemonstrated the desirability of seed that are homozygous for the trait.

We also noted the consequences of LC-PUFA synthesis on the overall fattyacid profile in these seed. Although we observed accumulation of new ω6and ω3 PUFA (i.e. products of Δ6-desaturation) at levels of greater than8% in DW5 seed, these seed had levels of the precursor fatty acids LAand ALA that were almost the same as in the wild-type seed. Rather thandepleting LA and ALA, the levels of monounsaturated fatty acid C18:1Δ⁹and its elongated derivatives (20:1Δ¹¹ and 22:1Δ¹³) were significantlyreduced. Thus it appeared that conversion of C₁₈-PUFA to LC-PUFAresulted in increased conversion of 18:1 to LA and ALA, and acorresponding reduction in 18:1 available for elongation.

The plant expression vector pXZP355 containing the Δ4-desaturase and theΔ5-elongase genes was also used to introduce the genes into plants ofthe homozygous line DO11-5, and 20 transgenic T1 plants were obtained.The levels of DHA and DPA in T2 seed from these plants were similar tothose observed in seed from DW5. Reductions in the levels of themonounsaturated fatty acids were also observed in these seed.

Fractionation of the total seed lipids of DW5 seed revealed them to becomprised of 89% TAG and 11% polar lipids (largely made upphospholipids). Furthermore, fatty acid analysis of the TAG fractionfrom DW5 seed showed that the newly synthesised EPA and DHA were beingincorporated into the seed oil and that the proportion of EPA and DHA inthe fatty acid composition of the total seed lipid essentially reflectedthat of the TAG fraction (Table 10).

Example 12 Isolation of Homologous Genes from Other Sources

Homologs of the desaturase and elongase genes such as the P. salinagenes described herein may be readily detected in other microalgae orother sources by hybridization to labelled probes derived from thegenes, particularly to parts or all of the coding regions, for exampleby Southern blot hybridization or dot-blot hybridisation methods. Thehomologous genes may be isolated from genomic or cDNA libraries of suchorganisms, or by PCR amplification using primers corresponding toconserved regions. Similarly, homologs of vertebrate desaturases withhigh affinity for Acyl-CoA and/or freshwater fish bifunctionaldesaturases can be isolated by similar means using probes to thezebrafish Δ5/Δ6 desaturase.

Dot Blot Hebridisations

Genomic DNA from six microalgae species was isolated using a DNAeasy kit(Qiagen) using the suppliers instructions, and used in dot blothybridization analyses for identification of homologous genes involvedin LC-PUFA synthesis in these species. This also allowed evaluation ofthe sequence divergence of such genes compared to those isolated fromPavlova salina. The species of microalga examined in this analysis werefrom the genera Melosira, Rhodomonas, Heterosigma, Nannochloropsis,Heterocapsa and Tetraselmis. They were identified according to Hasle, G.R. & Syvertsen, E. E. 1996 Dinoflagellates. In: Tomas, C. R. (ed.)Identifying Marine Phytoplankton. Academic Press, San Diego, Calif. pp531-532. These microalga were included in the analysis on the basis ofthe presence of EPA, DHA, or both when cultured in vitro (Example 2).

Genomic DNA (approximately 100 μg) isolated from each of the microalgawas spotted onto strips of Hybond N+ membrane (Amersham). After airdrying, each membrane strip was placed on a layer of 3 MM filter papersaturated with 0.4 M NaOH for 20 min, for denaturation of the DNA, andthen rinsed briefly in 2×SSC solution. The membrane strips were airdried and the DNA cross linked to the membranes under UV light. Probeslabeled with ³²P nucleotides and consisting of the coding regionswithout the untranslated regions of a number of Pavlova-derived genes,including the Δ8, Δ5 and Δ4 desaturases and Δ9 and Δ5 elongases, wereprepared and hybridized to each membrane strip/DNA dot blot. Themembranes were hybridized with each probe overnight in a buffercontaining 50 mM Tris-HCl, pH7.5, 1M NaCl, 50% formamide, 10×Denhardt'ssolution, 10% dextran sulfate, 1% SDS, 0.1% sodium pyrophosphate, and0.1 mg/ml herring sperm DNA, at 42° C., then washed three times in asolution containing 2×SSC, 0.5% SDS at 50° C. for 15 min each (lowstringency wash in this experiment) or for a high stringency wash in0.2×SSC, 0.5% SDS at 65° C. for 20 minutes each.

It is well understood that the stringency of the washing conditionsemployed in DNA blot/hybridizations can reveal useful informationregarding the sequence relatedness of genes. Thus hybridizationsmaintained when subjected to a high stringency wash indicate a highlevel of sequence relatedness (e.g. 80% or greater nucleotide identityover at least 100-200 nucleotides), while hybridizations maintained onlyduring low stringency washes indicate a relatively lower degree of DNAconservation between genes (e.g. 60% or greater nucleotide identity overat least 200 nucleotides).

The hybridized dot blots were exposed to BioMax X-ray film (Kodak), andthe autoradiograms are shown in FIG. 9. The autoradiograms reveal thepresence of homologs to the P. salina LC-PUFA genes in these species,and moreover reveal a range of homologies based on the different levelsof hybridization seen under the high and low stringency conditions. Itappeared that some of the microalgal species examined have LC-PUFA genesthat may differ substantially from the genes in P. salina, while othersare more related in sequence. For example, genes from Tetraseimis spappeared to be highly similar to the Δ4- and Δ5-desaturases and the Δ5elongase from Pavlova salina on the basis of the strength ofhybridizations. In contrast, all of the LC-PUFA genes identified inMelosira sp appeared to have lower degrees of similarity to the P.salina genes.

Isolation of an LC-PUFA Elongase Gene from Heterocapsa sp.

Heterocapsa spp. such as Heterocapsa niei in the CSIRO collection(Example 2) are dinoflagellates that were identified as producers ofLC-PUFA including EPA and DHA. To exemplify the isolation of LC-PUFAsynthesis genes from these dinoflagellates, DNA was purified from cellsof a Heterocapsa niei strain originally isolated in Port Hacking, NSW,Australia in 1977, DNA was isolated using a DNAeasy kit (Qiagen) usingthe suppliers instructions. Based on published multiple amino acidsequence alignments for fatty acid elongases (Qi et al., 2002;Parker-Barnes et al., 2000), the consensus amino acid blocks FLHXYH (SEQID NO:48) and MYXYYF (SEQ ID NO:49) were identified and correspondingdegenerate primers encoding these sequences5′-CAGGATCCTTYYTNCATNNNTAYCA-3′ (SEQ ID NO:50) (sense) or complementaryto these sequences 5′-GATCTAGARAARTARTANNNRTACAT-3′ (SEQ ID NO:51)(antisense) were synthesised. PCR amplification reactions were carriedout in reaction volumes of 20 μL with 20 pmol of each primer, 200 ng ofHeterocapsa sp. genomic DNA and Hotstar Taq DNA polymerase (Qiagen) withbuffer and nucleotide components as specified by the supplier. Thereactions were cycled as follows: 1 cycle of 95° C. for 15 minutes, 5cycles of 95° C., 1 min, 38° C., 1 min, 72° C., 1 min, 35 cycles of 95°C., 35 sec, 52° C., 30 sec, 72° C., 1 min, 1 cycle of 72° C., 10 min.Fragments of approximately 350 bp were generated and ligated intopGEM-Teasy for sequence analysis.

Of eight clones isolated, two identical clones had nucleotide andencoded amino acid sequences with similarity to regions of knownelongases. These were designated Het350Elo, and the nucleotide and aminoacid sequences are provided as SEQ ID NO:79 and SEQ ID NO:80respectively. BLAST analysis and the presence of an in-frame stop codonsuggested the presence of an intron between approximate positions 33 and211.

The best matches to the amino acid sequence were animal elongasesequences, see for example Meyer et al. (2004), indicating that theisolated Heterocapsa gene sequence was probably involved in elongationof C18 and C20 fatty acid substrates.

Full-length clones of the elongase can readily be isolated by screeninga Heterocapsa cDNA library or by 5′- and 3′ RACE techniques, well knownin the art.

Construction of Melosira sp. cDNA Library and EST Sequencing

mRNA, for the construction of a cDNA library, was isolated from Melosirasp. cells using the following method. 2 g (wet weight) of Melosira sp.cells were powdered using a mortar and pestle in liquid nitrogen andsprinkled slowly into a beaker containing 22 ml of extraction bufferthat was being stirred constantly. To this, 5% insolublepolyvinylpyrrolidone, 90 mM 2-mercaptoethanol, and 10 mM dithiotheitolwere added and the mixture stirred for a further 10 minutes prior tobeing transferred to a Corex™ tube. 18.4 ml of 3M ammonium acetate wasadded and mixed well. The sample was then centrifuged at 6000×g for 20minutes at 4° C. The supernatant was transferred to a new tube andnucleic acid precipitated by the addition of 0.1 volume of 3M NaAc (pH5.2) and 0.5 volume of cold isopropanol. After 1 hour incubation at −20°C., the sample was centrifuged at 6000×g for 30 minutes in a swing-outrotor. The pellet was resuspended in ml of water and extracted withphenol/chloroform. The aqueous layer was transferred to a new tube andnucleic acids were precipitated once again by the addition of 0.1 volume3M NaAc (pH 5.2) and 2.5 volume of ice cold ethanol. The pellet wasresuspended in water, the concentration of nucleic acid determined andthen mRNA was isolated using the Oligotex mRNA system (Qiagen).

First strand cDNA was synthesised using an oligo(dT) linker-primersupplied with the ZAP-cDNA synthesis kit (Stratagene—cat #200400) andthe reverse transcriptase SuperscriptIII (Invitrogen). Double strandedcDNA was ligated to EcoRI adaptors and from this a library wasconstructed using the ZAP-cDNA synthesis kit as described in theaccompanying instruction manual (Stratagene—cat #200400). A primarylibrary of 1.4×10⁶ plaque forming units (pfu) was obtained. The averageinsert size of cDNA inserts in the library was 0.9 kilobases based on 47random plaques and the percentage of recombinants in the library was99%.

Single pass nucleotide sequencing of 8684 expressed sequence tags (ESTs)was performed with SK primer (5′-CGCTCTAGAACTAGTGGATC-3′) (SEQ ID NO:87)using the ABI BigDye system. Sequences of 6750 ESTs were longer than 400nucleotides, showing the inserts were at least this size. ESTs showinghomology to several fatty acid desaturases and one PUFA elongase wereidentified by BlastX analysis.

The amino acid sequence (partial) (SEQ ID NO:88) encoded by the cDNAclone Mm301461 showed 75% identity to Thalassiosira pseudonana fattyacid elongase 1 (Accession No. AY591337). The nucleotide sequence of ESTclone Mm301461 is provided as SEQ ID NO:89. The high degree of identityto a known elongase makes it highly likely that Mm301461 encodes aMelosira fatty acid elongase. RACE techniques can readily be utilized toisolate the full-length clone encoding the elongase.

Example 13 Isolation of FAE-Like Elongase Gene Fragment from P. salina

Random cDNA clones from the P. salina cDNA library were sequenced by anEST approach. In an initial round of sequencing, 73 clones weresequenced. One clone, designated 11.B1, was identified as encoding aprotein (partial sequence) having sequence similarity with known betaketo-acyl synthase-like fatty acid elongases, based on BLASTX analysis.The nucleotide sequence of 11.B1 from the 3′ end is provided as (SEQ IDNO:55).

These plant elongases are different to the ELO class elongase in thatthey are known to be involved in the elongation of C16 to C18 fattyacids and also the elongation of very-long-chain saturated andmonounsaturated fatty acids. Clone 11.B1, represents the firstnon-higher plant gene in this class isolated.

Example 14 Isolation of a Gene Encoding a Δ5-Desaturase from P. salina

Isolation of a Gene Fragment from a Δ5-Desaturase Gene from P. salina

In order to isolate a Δ5-desaturase gene from P. salina,oligonucleotides were designed for a conserved region of desaturases.The oligonucleotides designated d5A and d5B shown below were madecorresponding to a short DNA sequence from a Δ5-desaturase gene fromPavlova lutheri. Oligo d5A:5′-TGGGTTGAGTACTCGGCCAACCACACGACCAACTGCGCGCCCTCGTGGTGGTGCGACTGGTGGATGTCTTACCTCAACTACCAGATCGAGCATCATCTGT-3′ (nucleotides 115-214of International patent application published as WO03078639-A2, FIG. 4a) (SEQ ID NO:56) and oligo d5B:5′-ATAGTGCAGCCCGTGCTTCTCGAAGAGCGCCTTGACGCGCGGCGCGATCGTCGGGTGGCGGAATTGCGGCATGGACGGGAACAGATGATGCTCGATCTGG-3′ (corresponding tothe complement of nucleotides 195-294 of WO03078639-A2, FIG. 4 a) (SEQID NO:57). These oligonucleotides were annealed and extended in a PCRreaction. The PCR product from was inserted into pGEM-T Easy vector andthe nucleotide sequence confirmed.

The cloned fragment was labelled and used as a hybridization probe forscreening of a Pavlova salina cDNA library under moderately highstringency conditions, hybridizing at 55° C. overnight with an SSChybridization solution and washing the blots at 60° C. with 2×SSC/0.1%SDS three times each for 10 minutes. From screening of about 500,000plaques, 60 plaques were isolated which gave at least a weakhybridization signal. Among 13 clones that were sequenced, one clonedesignated p1918 contained a partial-length cDNA encoding an amino acidsequence with homology to known Δ5-desaturase genes. For example, theamino acid sequence was 53% identical to amino acid residues 210-430from the C-terminal region of a Thraustochytrium Δ5-desaturase gene(Accession No. AF489588).

Isolation of a Full-Length Δ5-Desaturase Gene

The partial-length sequence in p1918 was used to design a pair ofsequence specific primers, which were then used in PCR screening of the60 isolated plaques mentioned above. Nineteen of the 60 were positive,having the same or similar cDNA sequence. One of the clones that showeda strong hybridization signal using the partial-length sequence as aprobe was used to determine the full-length sequence provided as SEQ IDNO:58, and the amino acid sequence (425 amino acids in length) encodedthereby is provided as SEQ ID NO:60.

The amino acid sequence was used to search the NCBI protein sequencedatabase using the BLASTX software. The results indicated that thissequence was homologous to known Δ5-desaturases. The amino acid sequenceof the P. salina protein showed 81% identity to a P. lutheri sequence ofundefined activity in WO03/078639-A2, and 50% identity to aΔ5-desaturase from Thraustochytrium (Accession No. AF489588). ThePavlova salina Δ5-desaturase showed all the conserved motifs typical of‘front-end desaturases’ including the N-terminal cytochrome b₅-likedomain and three conserved histidine-rich motifs.

Co-Expression of Δ9 Elongase, Δ8-Desaturase and Δ5-Desaturase Genes inTransformed Cells

Co-expression of the Δ5-desaturase gene together with the Δ9 elongasegene (Elo2, Example 7) and the Δ8-desaturase gene (Example 6) wasachieved in cells as follows. The plant expression vector pXZP354containing the three genes, each from P. salina, and each expressed fromthe seed specific napin promoter was constructed. The P. salinaΔ8-desaturase coding region from the cDNA clone (above) was firstinserted as a BamHI-NcoI fragment into pXZP143 between the seed specificnapin promoter and Nos terminator, resulting in plasmid pXZP46. The P.salina Δ9-elongase gene was likewise inserted, as a PstI-XhoI fragmentfrom its cDNA clone, into pXZP143 resulting in plasmid pXZP143-Elo2. TheP. salina Δ5-desaturase gene was also inserted, as a PstI-BssHIIfragment from its cDNA clone, into pXZP143, resulting in plasmidpXZP147. Then, the HindIII-ApaI fragment containing the Δ9-elongaseexpression cassette from pXZP143-Elo2 was inserted into pXZP146downstream of the Δ8-desaturase expression cassette, resulting inplasmid pXZP148. The HindIII-ApaI fragment containing the Δ5-desaturaseexpression cassette from pXZP147 was inserted into pXZP148 downstream ofΔ8-desaturase and Δ9-elongase expression cassettes, resulting in plasmidpXZP149. Then, as a final step, the HindIII-ApaI fragment containing thethree genes from pXZP149 was inserted into a derivative of the binaryvector pART27, containing a hygromycin resistance gene selection marker,resulting in plant expression plasmid pXZP354.

Plasmid pXZP354 was introduced into Arabidopsis by theAgrobacterium-mediated floral dip method, either in the simultaneouspresence or the absence of expression plasmid pXZP355 (Example 11)containing the P. salina Δ5-elongase and Δ4-desaturase genes.Co-transformation of the vectors could be achieved since they containeddifferent selectable marker genes. In the latter case, the transgenicplants (designated “DR” plants) were selected using hygromycin asselective agent, while in the former case, the plants (“DU” plants) wereselected with both hygromycin and kanamycin.

Twenty-one DR plants (T1 plants) were obtained. Fatty acid analysis ofseedoil from T2 seed from ten of these plants showed the presence of lowlevels of 20:2ω (EDA), 20:3ω6 (DGLA) and 20:4ω6 (ARA), including up to0.4% ARA. Fatty acid analysis of seedoil from T2 seed from seven DUplants showed similar levels of these fatty acids. From the relativeratios of these fatty acids, it was concluded that the Δ5-desaturase andΔ8-desaturase genes were functioning efficiently in seed transformedwith pXZP354 but that the activity of the Δ9 elongase gene wassuboptimal. It is likely that shortening of the coding region at theN-terminal end, to initiate translation at amino acid position 11 or 29of SEQ ID NO:3 (Example 9) (see SEQ ID NO's 85 and 86) will improve thelevel of activity of the Δ9 elongase gene. Expression of one or two ofthe genes from seed-specific promoters other than the napin promoter, sothey are not all expressed from the napin promoter, is also expected toimprove the expression level of the Δ9 elongase gene.

Example 15 Isolation of a Gene Encoding a Δ6-Desaturase from Echiumplantagineum

Some plant species such as evening primrose (Oenothera biennis), commonborage (Borago officinalis), blackcurrant (Ribes nigrum), and someEchium species belonging to the Boragenacae family contain the ω6- andω3-esaturated C18 fatty acids, γ-linolenic acid (18:3ω6, GLA) andstearidonic acid (18:4ω3, SDA) in their leaf lipids and seed TAGs(Gull-Guerrero et al., 2000). GLA and SDA are recognized as beneficialfatty acids in human nutrition. The first step in the synthesis ofLC-PUFA is a Δ6-desaturation. GLA is synthesized by a Δ6-desaturase thatintroduces a double bond into the Δ6-position of LA. The same enzyme isalso able to introduce a double bond into Δ6-position of ALA, producingSDA. Δ6-Desaturase genes have been cloned from members of theBoraginacae, like borage (Sayanova et al., 1997) and two Echium species(Garcia-Maroto et al., 2002).

Echium plantagineum is a winter annual native to Mediterranean Europeand North Africa. Its seed oil is unusual in that it has a unique ratioof ω3 and ω6 fatty acids and contains high amounts of GLA (9.2%) and SDA(12.9%) (Guil-Guerrero et al., 2000), suggesting the presence ofΔ6-desaturase activity involved in desaturation of both ω3 and ω6 fattyacids in seeds of this plant.

Cloning of E. platangineum EplD6Des Gene

Degenerate primers with built-in XbaI or SacI restriction sitescorresponding to N- and C-termini amino acid sequences MANAIKKY (SEQ IDNO: 61) and EALNTHG (SEQ ID NO: 62) of known Echium pitardii and Echiumgentianoides (Garcia-Maroto et al., 2002) Δ6-desaturases were used forRT-PCR amplification of Δ6-desaturase sequences from E. platangineumusing a proofreading DNA polymerase Pfu Turbo® (Stratagene). The 1.35 kbPCR amplification product was inserted into pBluescript SK(+) at theXbaI and SacI sites to generate plasmid pXZP106. The nucleotide sequenceof the insert was determined (SEQ ID NO:63). It comprised an openreading frame encoding a polypeptide of 438 amino acid residues (SEQ IDNO:64) which had a high degree of homology with other reported Δ6- andΔ8-desaturases from E. gentianoides (SEQ ID NO:65), E. pitardii (SEQ IDNO:66), Borago officinalis (SEQ ID NO:67 and 68). Helianthus annuus (SEQID NO:69) and Arabidopsis thaliana (SEQ ID NO:70 and SEQ ID NO:71) (FIG.10). It has a cytochrome b₅ domain at the N-terminus, including the HPGG(SEQ ID NO:72) motif in the heme-binding region, as reported for otherΔ6- and Δ8-desaturases (Sayanova et al. 1997; Napier et al. 1999). Inaddition, the E. plantagineum Δ6 desaturase contains three conservedhistidine boxes, including the third histidine box containing thesignature QXXHH (SEQ ID NO:73) motif present in majority of the‘front-end’ desaturases (FIG. 10) (Napier et al., 1999). Clusteranalysis including representative members of Δ6 and Δ8 desaturasesshowed a clear grouping of the cloned gene with other Δ6 desaturasesespecially those from Echium species.

Heterologous Expression of E. plantagineum Δ6-DESATURASE Gene in Yeast

Expression experiments in yeast were carried out to confirm that thecloned E. platangineum gene encoded a Δ6-desaturase enzyme. The genefragment was inserted as an XbaI-SacI fragment into the SmaI-SacI sitesof the yeast expression vector pSOS (Stratagene) containing theconstitutive ADH1 promoter, resulting in plasmid pXZP271. This wastransformed into yeast strain S288Cα by a heat shock method andtransformant colonies selected by plating on minimal media plates. Forthe analysis of enzyme activity, 2 mL yeast clonal cultures were grownto an O.D.₆₀₀ of 1.0 in yeast minimal medium in the presence of 0.1%NP-40 at 30° C. with shaking. Precursor free-fatty acids, eitherlinoleic or linolenic acid as 25 mM stocks in ethanol, were added sothat the final concentration of fatty acid was 0.5 mM. The cultures weretransferred to 20° C. and grown for 2-3 days with shaking. Yeast cellswere harvested by repeated centrifugation and washing first with 0.1%NP-40, then 0.05% NP-40 and finally with water. Fatty acids wereextracted and analyzed. The peak identities of fatty acids wereconfirmed by GC-MS.

The transgenic yeast cells expressing the Echium EplD6Des were able toconvert LA and ALA to GLA and SDA, respectively. Around 2.9% of LA wasconverted to GLA and 2.3% of ALA was converted to SDA, confirming theΔ6-desaturase activity encoded by the cloned gene.

Functional Expression of E. platangineum Δ6-Desaturase Gene inTransgenic Tobacco

In order to demonstrate that the EplD6Des gene could confer thesynthesis of Δ6 desaturated fatty acids in transgenic plants, the genewas expressed in tobacco plants. To do this, the gene fragment wasexcised from pXZP106 as an XbaI-SacI fragment and cloned into the plantexpression vector pBI121 (Clonetech) at the XbaI and SacI sites underthe control of a constitutive 35S CaMV promoter, to generate plantexpression plasmid pXZP341. This was introduced into Agrobacteriumtumefaciens AGL1, and used for transformation of tobacco W38 planttissue, by selection with kanamycin.

Northern blot hybridization analysis of transformed plants was carriedout to detect expression of the introduced gene, and total fatty acidspresent in leaf lipids of wild-type tobacco W38 and transformed tobaccoplants were analysed as described above. Untransformed plants containedappreciable amounts of LA (21% of total fatty acids) and ALA (37% oftotal fatty acids) in leaf lipids. As expected, neither GLA nor SDA,products of Δ6-desaturation, were detected in the untransformed leaf.Furthermore, transgenic tobacco plants transformed with the pBI121vector had similar leaf fatty acid composition to the untransformed W38plants. In contrast, leaves of transgenic tobacco plants expressing theEplD6Des gene showed the presence of additional peaks with retentiontimes corresponding to GLA and SDA. The identity of the GLA and SDApeaks were confirmed by GC-MS. Notably, leaf fatty acids of plantsexpressing the EplD6Des gene consistently contained approximately atwo-fold higher concentration of GLA than SDA even when the totalΔ6-desaturated fatty acids amounted up to 30% of total fatty acids intheir leaf lipids (Table 11).

TABLE 11 Fatty acid composition in lipid from transgenic tobacco leaves(%). Total Δ6- desaturate Plant 16:0 18:0 18:1 18:2 GLA 18:3 SDAproducts W38 21.78 5.50 2.44 21.21 — 37.62 — — ET27-1 20.33 1.98 1.2510.23 10.22 41.10 6.35 16.57 ET27-2 18.03 1.79 1.58 14.42 1.47 53.850.48 1.95 ET27-4 19.87 1.90 1.35 7.60 20.68 29.38 9.38 30.07 ET27-515.43 2.38 3.24 11.00 0.84 49.60 0.51 1.35 ET27-6 19.85 2.05 1.35 11.124.54 50.45 2.19 6.73 ET27-8 19.87 2.86 2.55 11.71 17.02 27.76 7.76 24.78ET27-11 17.78 3.40 2.24 12.62 1.11 51.56 0.21 1.32 ET27-12 16.84 2.161.75 13.49 2.71 50.80 1.15 3.86

Northern analysis of multiple independent transgenic tobacco linesshowed variable levels of the EplD6Des transcript which generallycorrelated with the levels of Δ6-desaturated products synthesized in theplants. For example, transgenic plant ET27-2 which contained low levelsof the EplD6Des transcript synthesised only 1.95% of its total leaflipids as Δ6-desaturated fatty acids. On the other hand, transgenicplant ET27-4 contained significantly higher levels of EplD6Destranscript and also had a much higher proportion (30%) of Δ6-desaturatedfatty acids in its leaf lipids.

Analysis of the individual tobacco plants showed that, withoutexception, GLA was present at a higher concentration than SDA eventhough a higher concentration of ALA than LA was present inuntransformed plants. In contrast, expression of EplD6Des in yeast hadresulted in approximately equivalent levels of conversion of LA into GLAand ALA into SDA. Echium plantagineum seeds, on the other hand, containhigher levels of SDA than GLA. EplD6Des probably carries out itsdesaturation in vivo in Echium plantagineum seeds on LA and ALAesterified to phosphatidyl choline (PC) (Jones and Harwood 1980). In thetobacco leaf assay, the enzyme is most likely desaturating LA and ALAesterified to the chloroplast lipid monogalactosyldiacylglyerol (MGDG)(Browse and Slack, 1981). In the yeast assay, free fatty acid precursorsLA and ALA added to the medium most likely enter the acyl-CoA pool andare available to be acted upon by EplD6Des in this form.

Functional Expression of E. platangineum Δ6-Desaturase Gene inTransgenic Seed

To show seed-specific expression of the Echium Δ6-desaturase gene, thecoding region was inserted into the seed-specific expression cassette asfollows. An NcoI-SacI fragment including the Δ6-desaturase coding regionwas inserted into pXZP6, a pBluescriptSK derivative containing a Nosterminator, resulting in plasmid pXZP157. The SmaI-ApaI fragmentcontaining the coding region and terminator EplD6Des-NosT was clonedinto pWVec8-Fp1 downstream of the Fp1 prompter, resulting in plasmidpXZP345. The plasmid pXZP345 was used for transforming wild typeArabidopsis plants, ecotype Columbia, and transgenic plants selected byhygromycin B selection. The transgenic plants transformed with this genewere designated “DP” plants.

Fatty acid composition analysis of the seed oil from T2 seed from elevenT1 plants transformed with the construct showed the presence of GLA andSDA in all of the lines, with levels of Δ6-desaturation productsreaching to at least 11% (Table 12). This demonstrated the efficientΔ6-desaturation of LA and ALA in the seed.

TABLE 12 Fatty acid composition in transgenic Arabiclopsis seedsexpressing Δ6-desaturase from Echium. Fatty acid Total Δ6- (%)desaturation Plant 18:2^(Δ9,12) 18:3^(Δ6,9,12) 18:3^(Δ9,12,15)18:4^(Δ6,9,12,15) products Columbia 16:0 18:0 18:1^(Δ9) (LA) (GLA) (ALA)(SDA) 20:0 20:1 (%) DP-2 8.0 2.8 22.9 27.3 2.5 11.3 0.7 1.6 15.8 3.2DP-3 7.8 2.7 20.6 25.9 3.0 12.1 0.8 1.7 17.8 3.8 DP-4 7.8 2.8 20.4 28.51.2 13.7 0.4 1.7 16.1 1.5 DP-5 8.2 3.2 17.4 29.3 1.2 14.2 0.3 2.1 15.61.6 DP-7 8.2 2.9 18.4 26.7 5.0 12.7 1.4 1.7 15.2 6.4 DP-11 9.0 3.5 17.828.4 3.0 13.4 0.9 2.1 13.9 3.8 DP-12 8.6 3.0 18.9 27.8 3.3 12.6 1.0 1.815.4 4.3 DP-13 8.7 2.9 14.4 27.3 8.5 13.7 2.6 1.7 12.4 11.1 DP-14 9.32.9 14.2 32.3 2.1 15.4 0.7 1.8 12.8 2.8 DP-15 8.2 2.9 17.8 30.1 0.3 15.30.2 1.9 15.5 0.5 DP-16 8.0 2.8 19.5 29.2 2.7 13.1 0.8 1.7 14.2 3.5

Example 16 Mutagenesis of E. platangineum EplD6Des Gene

To determine whether variability could be introduced into theΔ6-desaturase gene and yet retain desaturase activity, the E.platangineum Δ6-desaturase cDNA was randomly mutated by PCR using Taqpolymerase and EPD6DesF1 and EPD6DesR1 primers in the presence of dITPas described by Zhou and Christie (1997). The PCR products were clonedas XbaI-SacI fragments in pBluescript SK(+) at XbaI and SacI sites, andsequences of randomly selected clones determined. Random variants withamino acid residue changes were chosen to clone as XbaI-SacI fragmentsinto pBI121 and the enzyme activities of proteins expressed from thesevariants characterized in transgenic tobacco leaves as described abovefor the wild-type gene.

FIG. 11A represents the activity of the EplD6Des sequence variants whenexpressed in tobacco plants. The variants could be divided into twobroad classes in terms of their ability to carry out Δ6-desaturation.Mutations represented as empty diamonds showed substantial reductions inthe Δ6-desaturation activity while mutations denoted as solid diamondshad little or no effect on the activity of the encoded Δ6-desaturaseenzyme. FIG. 11B represents the quantitative effect that a selection ofmutations in the EplD6Des gene had on the Δ6-desaturase activity. AnL14P mutation in the cytochrome b₅ domain and an S301P mutation betweenhistidine box II and histidine box III of EplD6Des caused substantialreductions in their Δ6-desaturase activities, resulting in a 3- to5-fold reduction in total Δ6-desaturated fatty acids when compared tothe wild-type enzyme in W38 plants. Surprisingly, significant activitywas retained for each. In contrast, most of the variants examined, asexemplified by the S205N mutation, had no effect on the Δ6-desaturationactivity of EplD6Des gene.

Example 17 Comparison of acyl-CoA and acyl-PC Substrate DependentDesaturases for Production of LC-PUFA in Cells

As described above, the synthesis of LC-PUFA such as EPA and DHA incells by the conventional Δ6 desaturation pathway requires thesequential action of PUFA desaturases and elongases, shown schematicallyin FIG. 12 part A. This conventional pathway operates in algae, mosses,fungi, diatoms, nematodes and some freshwater fish (Sayanova and Napier,2004). The PUFA desaturases from algae, fungi, mosses and worms areselective for desaturation of fatty acids esterified to the sn-2position of phosphatidylcholine (PC) while the PUFA elongases act onfatty acids in the form of acyl-CoA substrates represented in theacyl-CoA pool in the endoplasmic reticulum (ER), which isphysiologically separated from the PC component of the ER. Therefore,sequentially desaturation and elongation reactions on a fatty acidsubstrate requires that the fatty acid is transferred between theacyl-PC and acyl-CoA pools in the ER. This requires acyltransferasesthat are able to accommodate LC-PUFA substrates. This “substrateswitching” requirement may account for the low efficiency observed inearlier reported attempts to re-constitute LC-PUFA biosynthesis(Beaudoin et al., 2000, Domergue et al., 2003a). The alternative Δ5desaturation pathway (FIG. 12 part B) suffers from the same disadvantageof requiring “substrate switching”.

As described in Example 5, the strategy of using a vertebrate desaturasewhich was able to desaturate acyl-CoA substrates, provided relativelyefficient production of LC-PUFA in plant cells including in seed. InExample 5, the combination of a Δ5/Δ6 desaturase from zebra fish with aΔ6 elongase from C. elegans had the advantage that both the desaturaseand the elongase enzymes had activity on acyl-CoA substrates in theacyl-CoA pool. This may explain why this strategy was more efficient inthe synthesis of LC-PUFA. To provide a direct comparison of the relativeefficiencies of using an acyl-CoA substrate-dependent desaturasecompared to an acyl-PC substrate-dependent desaturase, we conducted thefollowing experiment. This compared the use of the Echium Δ6 desaturase(Example 15) and the P. salina Δ5 desaturase (Example 14), both of whichare thought to use acyl-PC substrates, with the zebrafish Δ6/Δ5desaturase which uses an acyl-CoA substrate (Example 5).

A construct was prepared containing two acyl-PC dependent desaturases,namely the Echium Δ6 desaturase and P. salina Δ5 desaturase, incombination with the C. elegans Δ6 elongase. The Echium Δ6 desaturasegene on an NcoI-SacI fragment was inserted into pXZP143 (Example 15)resulting in pXZP192. The C. elegans Δ6 elongase gene (Fp1-CeElo-NosTexpression cassette) on the HindIII-ApaI fragment of pCeloPWVec8(Example 5) was inserted into the StuI-ApaI sites of pXZP147 (Example14) to make pXZP193. The HindIII-ApaI fragment of pXZP193 containingboth genes (Fp1-PsD5Des-NosT and Fp1-CeElo-NosT) was inserted into theApaI-StuI sites of pXZP192, resulting in plasmid pXZP194 containing thethree expression cassettes. The XbaI-ApaI fragment from pXZP194 wasinserted in a pWvec8 derivative, resulting in pXZP357.

The plasmid pXZP357 was used to transform plants of wild-typeArabidopsis ecotype Columbia by Agrobacterium-mediated floral dipmethod, and six transgenic plants were obtained after hygromycin B (20mg/L) selection. The transgenic T1 plants were designated “DT” plants.The hygromycin resistant transformed plants were transferred into soiland self-fertilised. The T2 seed were harvested and the seed fatty acidcomposition of two lines, DT1 and DT2, was analysed. The seed fattyacids of DT1 and DT2 contained low levels of 18:3ω6 and 18:4ω4 (0.9 and0.8% of GLA, 0.3% and 0.1% of SDA, respectively, Table 13). In addition,both DT1 and DT2 seed also contained 0.3% and 0.1% of the 20:4ω6 (ARA).However, there was no apparent synthesis of the ω3 fatty acid EPA ineither of the T2 seed lines, which probably reflected the greaterdesaturation ability of the Echium Δ6 desaturase on the ω6 substrate LAcompared to the ω3 substrate ALA (Example 15).

TABLE 13 Fatty acid composition of seed-oil from T2 seed of DT1 and DT2.Fatty acid values are % of total fatty acids. Fatty acid Control DT1 DT216:0 7.2 6.5 6.5 18:0 2.9 3.6 3.3 18:1ω9 20.0 23.2 22.3 18:2ω6 27.5 23.624.4 18:3ω3 15.1 15.4 16.1 20:0 2.2 2.0 1.9 20:1ω9/ω11 19.9 19.4 19.520:1ω7 2.2 3.4 3.0 20:2ω6 0.1 0.0 0.0 22:1ω7 0.0 0.0 0.0 Other minor 2.81.5 1.9 Total 100.0 98.6 98.9 New ω6-PUFA 18:3ω6 0.0 0.9 0.8 20:3ω6 0.00.0 0.0 20:4ω6 0.0 0.3 0.1 Total 0.0 1.2 0.9 New ω3-PUFA 18:4ω3 0.0 0.30.2 20:4ω3 0.0 0.0 0.0 20:5ω3 0.0 0.0 0.0 Total 0.0 0.3 0.2 Total fattyacids 100.0 100.0 100.0

This data was in clear contrast to Example 5, above, where expression ofthe acyl-CoA dependent desaturase from zebrafish in combination with aΔ6 elongase resulted in the production of at least 1.1% ARA and 2.3% EPAin T2 seed fatty acids. Thus it would appear that acyl-PC dependentdesaturases were less effective than acyl-CoA dependent desaturases indriving the synthesis of LC-PUFA in plants cells.

Example 18 Expression of LC-PUFA Genes in Synechococcus

Synechococcus spp. (Bacteria; Cyanobacteria; Chroococcales;Synechococcus species for example Synechococcus elongatus, also known asSynechocystis spp.) are unicellular, photosynthetic, marine orfreshwater bacteria in the order cyanobacteria that utilize chlorophylla in the light-harvesting apparatus. The species include importantprimary producers in the marine environment. One distinct biochemicalfeature of Synechococcus is the presence of phycoerythrin, an orangefluorescent compound that can be detected at an excitation wavelength of540 nm, and which can be used to identify Synechococcus. Members of themarine synechococccus group are closely related at the level of 16srRNA. They are obligately marine and have elevated growth requirementsfor Na⁺, Cl⁻, Mg²⁺, and Ca²⁺, but can be grown readily in both naturaland artificial seawater liquid media as well as on plates (Waterbury etal. 1988). Since they have a rapid heterotrophic or autotrophic growthrate, contain fatty acid precursors such as LA and ALA, and arerelatively simple to transform, they are suitable for functional studiesinvolving LC-PUFA synthesis genes, or for production of LC-PUFA infermenter type production systems. Strains such as Synechococcus sp.strain WH8102, PCC7002 (7002, marine), or PCC7942 (freshwater) can begrown easily and are amenable to biochemical and genetic manipulation(Carr, N. G., and N. H. Mann. 1994. The oceanic cyanobacterialpicoplankton, p. 27-48. In D. A. Bryant (ed.), the Molecular biology ofcyanobacteria. Kluwer Academic publishers, Boston). For example,Synechococcus has been used as a heterologous expression system fordesaturases (Domergue 2003b).

Wildtype Synechococcus 7002 Fatty Acid Profile and Growth Rates

To show that cyanobacterium Synechococcus 7002 was a suitable host forthe transformation of fatty acid synthesis genes and that thisexpression system could be used to rapidly test the functions andspecificities of fatty acid synthesis genes, the growth of the wildtypestrain 7002 was first analysed at 22° C., 25° C. and 30° C. and theresultant fatty acid profiles analysed by gas chromatography for growthat 22° C. and 30° C. (Table 14).

TABLE 14 Synechococcus 7002 wildtype fatty acid profiles at 22° C. and30° C. growth temperatures (% of total fatty acid). Temp MyristicPalmitic Palmitoleic Stearic Oleic 18:1iso Linoleic GLA Linolenic 22° C.0.79 42.5 10.6 0.92 8.4 1.5 7.5 0.54 27.1 30° C. 0.76 47.1 10.9 0.6717.0 0.34 20.4 2.9

Growth at 30° C. was much more rapid than at 22° C., with intermediaterates at 25° C. (FIG. 13). The cells were found to contain both linoleic(LA, 18:2 ω6) and linolenic (ALA, 18:3 ω3) acids which could be used asprecursors for LC-PUFA synthesis. Although some of the preferredprecursor ALA was produced at the 30° C., higher levels were obtained at22° C. Tests were also carried out to determine whether cells could begrown at 30° C., followed by reducing the incubation temperature to 22°C. after sufficient biomass had been achieved, to see if this wouldresult in a shift to higher production of linolenic acid (FIG. 14). Inthis experiment, levels of ALA obtained were greater than 5%. In furtherexperiments, 25° C. was used as the preferred temperature for strain7002, providing adequate growth rates and suitable precursor fatty acidprofile.

Transformation Strategy

Both replicative plasmid vectors and non-replicative homologousrecombination vectors have been used previously to transform variouscyanobacterial species, including Synechococcus 7002 (Williams andSzalay, 1983; Ikeda et al., 2002; Akiyama et al., 1998a). Therecombination vectors may be preferred in certain applications, and havebeen used to inactivate a gene, rather than create an expression strain.

A recombination vector was constructed that was suitable forintroduction of one or more fatty acid synthesis genes into thechromosome of Synechococcus strains such as strain 7002. This vectorcontained the Synechococcus 7002 sul2 gene in a pBluescript plasmidbackbone, which provided an ampicillin gene as a selectable marker andallowed bacterial replication in species such as E. coli. The vector wasengineered to contain a plat promoter from E. coli fused to a downstreammultiple cloning site, with the two elements inserted approximately inthe centre of the sul2 gene. The sul2 gene in Synechococcus encodes alow affinity sulfate which is not essential under normal growthconditions. Any gene other than sul2, preferably a non-essential gene,could have been chosen for incorporation in the recombination vector.

The sul2 gene was amplified from Synechococcus 7002 genomic DNA usinggene-specific primers, based on the near-identical sequence in strainPCC6803 (Genbank Accession No. NC_(—)000911, nucleotides 2902831 to2904501) and inserted into the vector pGEM-T. The plac promoter frompBluescript was amplified using the primers5′-gctacgcccggggatcctcgaggctggcgcaacgcaattaatgtga-3′ (SEQ ID NO:81)(sense) and5′-cacaggaaacagcttgacatcgattaccggcaattgtacggcggccgctacggatatcctcgctcgagctcgcccggggtagct-3′ (SEQ ED NO:82) (antisense), which also introduced a number ofrestriction sites at the ends of the promoter sequence. The amplifiedfragment was then digested with SmaI and ligated to the large PvuIIfragment of pBluescript including the beta-lactamase gene. Thisintermediate vector was then digested with EcoRV and SacI and ligated tothe HpaI to SacI fragment (designated sul2b) of the sul2 gene. Theresultant plasmid was digested with BamHI, treated with DNA polymerase I(Klenow fragment) to fill in the ends, and ligated to the SmaI to HpaIfragment (designated sul2a) of the sul2 gene. Excess restriction siteswere then removed from this vector by digestion with SacI and SpeI,blunting the ends with T4 DNA polymerase, and religation. Finally, amultiple cloning site was introduced downstream of the plac promoter bydigesting the vector with ClaI and NotI, and ligating in a ClaI to NotIfragment from pBluescript, generating the recombination vector which wasdesignated pJRP3.2.

Various genes related to LC-PUFA synthesis were adapted by PCR methodsto include flanking restriction sites as well as ribosome binding site(RBS) sequences that were suitable for expression in the prokaryote,Synechococcus. For example, the Echium plantagineum Δ6-desaturase(Example 15) was amplified with the primers5′-AGCACATCGATGAAGGAGATATACCCatggctaatgcaatcaagaa-3′ (SEQ ID NO:83)(sense) and 5′-ACGATGCGGCCGCTCAACCATGAGTATTAAGAGCTT-3′ (SEQ ID NO:84)(antisense).

The amplified product was digested with ClaI and Nod and cloned into theClaI to NotI sites of pJRP3.2. A selectable marker gene comprising achloramphenicol acetyl transferase coding region (CAT) (catB3 gene,Accession No. AAC53634) downstream of a pbsA promoter (psbA-CAT) wasinserted into the XhoI site of pJRP3.2, producing the vector pJRP3.3.The selectable marker gene was inserted within the sulB gene to enableeasy selection for homologous recombination events after introduction ofthe recombination vector into Synechococcus.

Transformation of Synechococcus 7002 was achieved by mixing vector DNAwith cells during the exponential phase of growth, during which DNAuptake occurred, as follows. Approximately 1 μg of the recombinationvector DNA resuspended in 100 μL of 10 mM Tris-HCl was added to 900 μLof mid-log phase cells growing in BG-11 broth. The cells were incubatedfor 90 min at 30° C. and light intensity of 20 μmol photons·m⁻²·s⁻¹. 250μL aliquots were then added to 2 mL BG-11 broth, mixed with 2 mL moltenagar (1.5%) and poured onto BG-11 agar plates containing 50 μg/mLchloramphenicol (Cm) for selection of recombinant cells. The plates wereincubated for 10-14 days at the same temperature/light conditions beforethe Cm-resistant colonies were clearly visible. These colonies were thenre-streaked several times onto fresh BG-11/Cm50 plates. After severalrounds of restreaking on selective plates, liquid medium was inoculatedwith individual colonies and the cultures incubated at 25° C.

Synechococcus 7002 cells containing the Echium Δ6-desaturase geneinserted into the sulB gene via the recombination vector and expressedfrom the plac promoter are shown to produce GLA (18:3 Δ6,9,12) and SDA(18:4, Δ6,9,12,15) from endogenous linoleic acid (LA) and linolenic acid(ALA), respectively, as substrates.

Episomal vectors can also be used in Synechococcus rather than theintegrative/recombinational vectors described above. Synechococcusspecies have native plasmids that have been adapted for use intransformation, for example pAQ-EX1, where a fragment of the nativeplasmid pAQ1 (Accession No. NC_(—)005025) was fused with an E. coliplasmid to form a shuttle vector with both E. coli and Synechococcusorigins of replication (Ikeda et al., 2002; Akiyama et al., 1998b).

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

All publications discussed above are incorporated herein in theirentirety.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed before the priority dateof each claim of this application.

REFERENCES

-   Abbadi, A. et al., (2001) Eur. J. Lipid. Sci. Technol. 103:106-113.-   Abbadi, A., et al. (2004) Plant Cell 16:2734-2748.-   Abbott et al., (1998) Science 282:2012-2018.-   Agaba, M. et al., (2004) Marine Biotechnol (NY) 6:251-261.-   Akiyama, H. et al. (1998a) DNA Res. 5:327-334.-   Akiyama, H. et al. (1998b) DNA Res. 5:127-129.-   Baumlein, H. et al., (1991) Mol. Gen. Genet. 225:459-467.-   Baumlein, H. et al., (1992) Plant J. 2:233-239.-   Beaudoin, F. et al., (2000) Proc. Natl. Acad. Sci U.S.A.    97:6421-6426.-   Berberich, T. et al., (1998) Plant Mol. Biol. 36:297-306.-   Bolch, C. J. et al., (1999a) J. Phycology 35:339-355.-   Botch, C. J. et al., (1999b) J. Phycology 35:356-367.-   Broun, P. et al., (1998) Plant J. 13:201-210.-   Brown, M. R. et al., (1997) Aquaculture 151:315-331,-   Browse. J. A. and Slack. C. R. (1981) FEBS Letters 131:111-114.-   Chinain, M. et al., (1997) J. Phycology 33:36-43.-   Cho, H. P. et al., (1999a) J. Biol. Chem. 274:471-477.-   Cho, H. P. et al., (19996) J. Biol Chem 274:37335-37339.-   Clough, S. J. and Bent, A. F. (1998) Plant J. 16:735-43.-   Coleman, A. W. (1977) Am. J. Bot, 64:361-368.-   Domergue, F. et al., (2002) Eur. J. Biochem. 269:4105-4113.-   Domergue, F. et al., (2003a) J. Biol. Chem. 278:35115-35126.-   Domergue, F. et al., (2003b) Plant Physiol. 131:1648-1660.-   Drexler, H. et al., (2003) J. Plant Physiol. 160:779-802.-   Dunstan, G. A. et al., (1994) Phytochemistry 35:155-161.-   Gallagher, J. C. (1980) J. Phycology 16:464-474.-   Garcia-Maroto, F. et al., (2002) Lipids 37:417-426.-   Girke, T. et al., (1998) Plant J. 15:39-48.-   Guil-Guerrero, J. L. et al., (2000). Phytochemistry 53:451-456.-   Haseloff, J. and Gerlach, W. L. (1988) Nature 334:585-591.-   Hastings, N. et al., (2001) Proc. Natl. Acad. Sci. U.S.A.    98:14304-14309.-   Hong, H. et al., (2002) Lipids 37:863-868.-   Hong, H. et al., (2002a) Lipids 37:863-868.-   Horiguchi, G. et al., (1998) Plant Cell Physiol. 39:540-544.-   Huang, Y. S. et al., (1999) Lipids 34:649-659.-   Ikeda, K. et al. (2002) World J. Microbiol. Biotech. 18:55-56.-   Inagaki, K. et al., (2002) Biosci Biotechnol Biochem 66:613-621.-   Jones, A. V. and Harwood, J. L (1980) Biochem J. 190:851-854.-   Kajikawa, M. et al., (2004) Plant Mol Biol 54:335-52.-   Knutzon, D. S. et al., (1998) J. Biol Chem. 273:29360-6.-   Lee, M. et al., (1998) Science 280:915-918.-   Leonard, A. E. et al., (2000) Biochem. J. 347:719-724.-   Leonard, A. E. et al., (2000b) Biochem. J. 350:765-770.-   Leonard, A. E. et al., (2002) Lipids 37:733-740.-   Lo, J. et al., (2003) Genome Res. 13:455-466.-   Mansour, M. P. et al., (1999a) J. Phycol. 35:710-720.-   Medlin, L. K. et al., (1996) J. Marine Systems 9:13-31.-   Metz, J. G. et al., (2001) Science 293:290-293.-   Meyer, A. et al., (2003) Biochemistry 42:9779-9788.-   Meyer, A. et al., (2004) Lipid Res 45:1899-1909.-   Michaelson. L. V. et al., (1998a) J. Biol. Chem. 273:19055-19059.-   Michaelson, L. V. et al., (1998b) FEBS Lett. 439:215-218.-   Mitchell, A. G. and Martin, C. E. (1995) J. Biol. Chem.    270:29766-29772.-   Morita, N. et al., (2000) Biochem. Soc. Trans. 28:872-879.-   Napier, J. A. et al., (1998) Biochem J. 330:611-614.-   Napier, J. A et. al., (1999) Trends in Plant Sci 4:2-4.-   Napier, J. A. et al., (1999) Curr. Op. Plant Biol. 2:123-127,-   Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48:443-453.-   Parker-Barnes, J. F. et al., (2000) Proc Natl Acad Sci USA    97:8284-8289.-   Pereira, S. L. et al., (2004) Biochem. J. 378:665-671.-   Perriman, R. et al., (1992) Gene 113:157-163.-   Qi, B. et al., (2002) FEBS Lett. 510:159-165.-   Qiu, X. et al., (2001) J. Biol. Chem. 276:31561-31566.-   Reddy, A. S. et al., (1993) Plant Mol. Biol. 22:293-300.-   Saito, T. et al., (2000) Eur. J. Biochem. 267:1813-1818.-   Sakuradani, E. et al., (1999) Gene 238:445-453.-   Sayanova, O. V. et al., (1997) Proc. Natl. Acad. Sci. U.S.A.    94:4211-4216.-   Sayanova, O. V. et al., (1999) Plant Physiol. 121:641-646.-   Sayanova, O. V. et al., (2003) FEBS Lett. 542:100-104.-   Sayanova, O. V. and Napier, J. A. (2004) Phytochemistry 65:147-158.-   Shippy, R. et al., (1999) Mol. Biotech. 12:117-129.-   Simopoulos, A. P. (2000) Poultry Science 79:961-970.-   Singh, S. et al., (2001) Planta 212:872-879.-   Smith, N. A. et al., (2000) Nature 407:319-320.-   Sperling, P. et al., (2000) Eur. J. Biochem. 267:3801-3811.-   Sperling, P. and Heinz, E. (2001) Eur. J. Lipid Sci. Technol    103:158-180,-   Sprecher, H. et al., (1995) J. Lipid Res. 36:2471-2477.-   Spychalla, P. J. et al., (1997) Proc. Natl. Acad. Sci. U.S.A.    94:1142-1147.-   Stalberg, K. et al., (1993) Plant. Mol. Biol. 23:671-683.-   Takeyama, H. et al., (1997) Microbiology 143:2725-2731.-   Tanaka, M. et al., (1999) Biotechnol. Lett. 21:939-945.-   Tonon, T. et al., (2003) FEBS Lett. 553:440-444.-   Trautwein, E. A. (2001) Eur. J. Lipid Sci. Technol. 103:45-55.-   Tvrdik, P. (2000) J. Cell Biol. 149:707-718.-   Valvekens, D. et al., (1988) Proc. Natl. Acad. Sci. USA.    85:5536-5540.-   Volkman, J. K. et al., (1989) J. Exp. Mar. Biol. Ecol. 128:219-240.-   Wallis, J. G. and Browse, J. (1999) Arch. Biochem. Biophys.    365:307-316.-   Wang, M. B. et al., (1997) J. Gen. Breed. 51:325-334.-   Waterbury, J. B. et al. (1988) Methods Enzymol. 167:100-105.-   Waterhouse, P. M. et al., (1998) Proc. Natl. Acad. Sci. USA    95:13959-13964.-   Watts, J. L. and Browse, J. (1999b) Arch Biochem Biophys    362:175-182.-   Williams, J. G. and Szalay, A. A. (1983) Gene 24:37-51,-   Whitney, H. M. et al., (2003). Planta 217:983-992.-   Yazawa, K. (1996) Lipids 31:5297-S300.-   Yu, R. et al., (2000) Lipids 35:1061-1064.-   Zank, T. K. et al., (2000) Plant J. 31:255-268.-   Zank, T. K. et al., (2002) Plant J. 31:255-268.-   Zhang, Q. et al., (2004) FEBS Lett. 556:81-85.-   Zhou, X. R. and Christie, P. J. (1997) J Bacterial 179:5835-5842.

The invention claimed is:
 1. A process for producing oil containingeicosapentaenoic acid and docosapentaenoic acid, comprising the steps ofobtaining a transgenic seed of an oilseed plant which compriseseicosapentaenoic acid and docosapentaenoic acid in an esterified form aspart of triglycerides, and a microalgal fatty acid desaturase, whereinthe total fatty acid of the transgenic seed comprises at least 2.5% ω3C20 fatty acids (w/w) and wherein the docosapentaenoic acid is presentat a level based on a conversion ratio of eicosapentaenoic acid todocosapentaenoic acid of at least 5% (w/w), and extracting oil from thetransgenic seed so as to thereby produce the oil.
 2. The process ofclaim 1, wherein the step of extracting the oil comprises crushing theseed.
 3. The process of claim 1, wherein the total fatty acid of theseed comprises at least 9% C20 fatty acids (w/w).
 4. The process ofclaim 1, wherein the total fatty acid of the seed comprises at least1.5% eicosapentaenoic acid and at least 0.13% docosapentaenoic acid(w/w).
 5. The process of claim 1, wherein the total fatty acid of theseed comprises at least 2.1% eicosapentaenoic acid and less than 0.1%eicosatrienoic acid (w/w).
 6. The process of claim 1, wherein at least25% (w/w) of the eicosapentaenoic acid and docosapentaenoic acid of theseed is incorporated into triacylglycerols in the seed.
 7. The processof claim 1, wherein at least 50% (w/w) of the eicosapentaenoic acid anddocosapentaenoic acid of the seed is incorporated into triacylglycerolsin the seed.
 8. The process of claim 1, wherein the oil comprises atleast 50% triacylglycerols.
 9. The process of claim 1, wherein the totalfatty acid of the seed comprises at least 7.9% C20 fatty acids (w/w).10. The process of claim 9, wherein the total fatty acid of the seedcomprises at least 10.2% C20 fatty acids (w/w).
 11. The process of claim10, wherein the total fatty acid of the seed comprises at least 1.5%eicosapentaenoic acid and at least 0.13% docosapentaenoic acid (w/w).12. The process of claim 10, wherein at least 50% (w/w) of theeicosapentaenoic acid and docosapentaenoic acid of the seed isincorporated into triacylglycerols in the seed.
 13. The process of claim1, wherein the docosapentaenoic acid is present at a level based on aconversion ratio of eicosapentaenoic acid to docosapentaenoic acid of atleast 7% (w/w).
 14. The process of claim 13, wherein the total fattyacid of the seed comprises at least 1.5% eicosapentaenoic acid and atleast 0.13% docosapentaenoic acid (w/w).
 15. The process of claim 13,wherein at least 50% (w/w) of the eicosapentaenoic acid anddocosapentaenoic acid of the seed is incorporated into triacylglycerolsin the seed.
 16. The process of claim 13, wherein the docosapentaenoicacid is present at a level based on a conversion ratio ofeicosapentaenoic acid to docosapentaenoic acid of at least 10% (w/w).17. The process of claim 16, wherein the total fatty acid of the seedcomprises at least 1.5% eicosapentaenoic acid and at least 0.13%docosapentaenoic acid (w/w).
 18. The process of claim 16, wherein atleast 50% (w/w) of the eicosapentaenoic acid and docosapentaenoic acidof the seed is incorporated into triacylglycerols in the seed.
 19. Theprocess of claim 1, wherein the transgenic seed is a transgenic canolaseed.
 20. The process of claim 4, wherein the transgenic seed is atransgenic canola seed.